Mutated genes for the catalytic protein of Oplophorus luciferase and use thereof

ABSTRACT

Secreted luciferases which are different from those known heretofore have been desired. The present invention provides a luciferase mutant comprising an amino acid sequence in which at least one amino acid selected from amino acids at the positions of 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This is a Divisional of copending application Ser. No. 15/157,930, filedMay 18, 2016, which is a Divisional of application Ser. No. 14/576,366,filed Dec. 19, 2014 (now U.S. Pat. No. 9,382,520, issued Jul. 5, 2016),which claims priority to Japanese Patent Application No. 2013-268416,filed Dec. 26, 2013, the entire contents of each of which areincorporated by reference herein in their entirety.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 19, 2016 isnamed 206313_0014_02_SL_ST25.txt, and is 45,601 bytes in size.

TECHNICAL FIELD

The present invention relates to mutated genes for the catalytic proteinof Oplophorus luciferase, use thereof and so on.

BACKGROUND OF INVENTION

Bioluminescence is a phenomenon based on a chemical reaction in vivo,which is called a luciferin (a luminescence substrate)-luciferase (anenzyme that catalyzes the luminescence reaction) reaction. Numerousstudies of the identification of luciferins or luciferases and theelucidation of the luminescence mechanism in a molecular level have beenperformed for a long time in the country and overseas. In bioluminescentmarine organisms, Oplophorus gracilirostris luciferase from the deep-seashrimp is an extracellularly secreted luciferase (Non-Patent Document1).

Oplophorus luciferase is a 106 kDa protein composed of a protein with amolecular weight of 35 kDa and a protein with a molecular weight of 19kDa. The domain that catalyzes the luminescence is found to be 19 kDaprotein. Oplophorus luciferase uses coelenterazine as a luminescencesubstrate and is classified as a coelenterazine-type luciferase (PatentDocument 1, Non-Patent Document 2). Oplophorus luciferase is differentfrom other coelenterazine-type luciferases in broad substratespecificity and uses coelenterazine analogues as a suitable substrate aswell as coelenterazine (Non-Patent Document 2). When the gene for the 19kDa protein is expressed in Escherichia coli (E. coli) at ambient andlower temperatures, the protein is expressed mostly as an insolubleprotein (Non-Patent Document 3). When the 19 kDa protein was expressedas a fusion protein to ZZ domain from protein A in a low temperatureexpression system, the fused protein could be expressed as a solubleprotein (Non-Patent Document 4). It is reported that when the 19 kDaprotein was expressed in animal cultured cells, the expressed proteinwas hardly secretion outside of cells (Non-Patent Document 2).

Recently, it is reported that the mutated 19 kDa protein havingcatalytic activity of luminescence was prepared by mutating the 16 aminoacids of the 19 kDa protein and showed higher luminescence activity thannative 19 kDa protein, and was secreted into an extracellular medium(Patent Document 2, Non-Patent Documents 4 and 5). It is also reportedthat coelenterazine derivatives displayed higher activity than nativecoelenterazine used as a substrate (Non-Patent Documents 4 and 5).

In the luminescence reaction system using coelenterazine as a substrate,the luminescence reaction of luciferase proceeds only by a substrate andmolecular oxygen. From this reason, a coelenterazine-type luciferasegene is used widely as a reporter assay in an animal cultured cellsystem at present. Renilla luciferase having 311 amino acids is used fora reporter assay inside of cells. For an extracellular reporter assay,the secreted Gaussia luciferase which is a secretory luciferase with asecretory signal peptide sequence of 17 amino acids and having 168 aminoacids is used.

RELATED ART DOCUMENTS

[Patent Documents]

[Patent Document 1] Japanese Laid-Open Patent Publication (Tokkai) No.2002-320482

[Patent Document 2] Japanese National Publication (Tokuhyo) No.2012-525819

[Non-Patent Documents]

[Non-Patent Document 1] O. Shimomura et al. (1978) Biochemistry 17:994-998.

[Non-Patent Document 2] S. Inouye et al. (2000) FEBS Lett. 481: 19-25.

[Non-Patent Document 3] S. Inouye & S. Sasaki (2007) Protein Express.Purif 56: 261-268.

[Non-Patent Document 4] M. P. Hall et al. (2012) ACS Chem Biol. 7:1848-1857.

[Non-Patent Document 5] S. Inouye et al. (2013) Biochem. Biophys. Res.Commun. 437: 23-28.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Under the foregoing circumstances, a novel luciferase that is distinctfrom conventional luciferase has been desired.

Means for Solving the Problem

The present inventors have made extensive investigations to solve theproblem above, and examined the reported 19 kDa protein mutants whichcatalyze the luminescence reaction. As a result, the inventors have anewly constructed luciferase mutant or the like, which are secretedextracellularly in the absence of any secretory signal peptidesequences, when the mutant is expressed in animal cultured cells. Thepresent invention has thus been accomplished.

More specifically, the present invention provides the followingluciferase mutants, polynucleotides, recombinant vectors, a method ofproducing luciferase mutants, kits, a method for performing aluminescence reaction, and so on.

[1] A luciferase mutant selected from (a) to (d) below:

(a) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from the 1st to 4th amino acids is deletedin the amino acid sequence of SEQ ID NO: 2;

(b) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 is anamino acid sequence in which 1 to 17 amino acids are deleted,substituted, inserted and/or added in the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;

(c) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 hasat least 90% identity to the amino acid sequence at the positions of 5to 169 of SEQ ID NO: 2, and having a luciferase activity; and,

(d) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 isencoded by a polynucleotide which hybridizes under stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa nucleotide sequence encoding the amino acid sequence at the positionsof 5 to 169 of SEQ ID NO: 2, and having a luciferase activity.

[2] The luciferase mutant according to [1] above, wherein the luciferasemutants defined in (b) to (d) above are mutants defined in (b-1) to(d-1) below:

(b-1) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 is anamino acid sequence in which 1 to 9 amino acids are deleted,substituted, inserted and/or added in the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;

(c-1) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 hasat least 95% identity to the amino acid sequence at the positions of 5to 169 of SEQ ID NO: 2, and having a luciferase activity; and,

(d-1) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 isencoded by a polynucleotide which hybridizes under high stringentconditions to a polynucleotide consisting of a nucleotide sequencecomplementary to a nucleotide sequence encoding the amino acid sequenceat the positions of 5 to 169 of SEQ ID NO: 2, and having a luciferaseactivity.

[3] The luciferase mutant according to [1] above, wherein the luciferasemutant of (a) above comprises the amino acid sequence selected from SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[4] A polynucleotide comprising a polynucleotide encoding the luciferasemutant according to any one of [1] to [3] above.

[5] A recombinant vector comprising the polynucleotide according to [4]above.

[6] A transformant transformed with the recombinant vector according to[5] above.

[7] A method of producing the luciferase mutant according to any one of[1] to [3] above, which comprises the steps of culturing thetransformant of [6] above and producing the luciferase mutant accordingto any one of [1] to [3] above.

[8] A kit comprising at least one selected from the luciferase mutantaccording to any one of [1] to [3] above, the polynucleotide accordingto [4] above, the recombinant vector according to [5] above and thetransformant according to [6] above.

[9] The kit according to [8] above, further comprising a luciferin.

[10] The kit according to [9] above, wherein the luciferin iscoelenterazines.

[11] The kit according to [10] above, wherein the coelenterazines arebis-coelenterazine or 6h-f-coelenterazine.

[12] A method for performing a luminescence reaction, which comprisescontacting the luciferase mutant according to any one of [1] to [3]above with a luciferin.

[13] The method according to [12] above, wherein the luciferin iscoelenterazines.

[14] The method according to [13] above, wherein the coelenterazines arebis-coelenterazine or 6h-f-coelenterazine.

[15] A method for assaying an activity of a sequence associated with theregulation of a promoter, which comprises using the polynucleotideaccording to [4] above as a reporter gene and contacting a luciferasemutant encoded by the reporter gene with a luciferin.

[16] The method according to [15] above, wherein the luciferin iscoelenterazines.

[17] The method according to [16] above, wherein the coelenterazines arebis-coelenterazine or 6h-f-coelenterazine.

[18] A method of visualizing a luminescence reaction, which comprisescontacting the luciferase mutant according to any one of [1] to [3]above with a luciferin.

[19] The method according to [18] above, wherein the luciferin iscoelenterazines.

[20] The method according to [19] above, wherein the coelenterazines areh-coelenterazine or f-coelenterazine.

Effects of the Invention

The present invention provides secreted luciferase mutants that aredistinct from the known mutants. In a preferred embodiment of theinvention, the luciferase mutants are novel luciferases which aresecreted extracellularly from the endoplasmic reticulum but not via thetrans-Golgi network when the proteins are expressed in animal cells, andcan be used to visualize the secretory pathway.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the results of SDS-PAGE analysis of the crude enzymesolution (crude extracts) of E. coli in which the amino-terminal deletednanoKAZ was expressed using a pColdII vector.

FIG. 2 shows the results of SDS-PAGE analysis of the crude enzymesolution of E. coli in which the amino-terminal deleted nanoKAZ wasexpressed using a pCold-ZZ-P vector.

FIG. 3 shows the secretory inhibition of nanoKAZ by brefeldin A from thestable expressed cell line, pcDNA3-GLsp-nanoKAZ/CHO-K1, which wasestablished using the nanoKAZ gene with a secretory signal peptidesequence. Open circles and closed circles indicate the presence andabsence of brefeldin A, respectively.

FIG. 4 shows the secretion of nanoKAZ from the stable expressed cellline, pcDNA3-nanoKAZ/CHO-K1, which was established using the nanoKAZgene lacking a secretory signal peptide sequence, in the presence ofbrefeldin A. Open circles and closed circles indicate the presence andabsence of brefeldin A, respectively.

FIG. 5 shows the bright-field image and the visualized luminescenceimage of nanoKAZ secretion. Labeled a and b represent the bright-fieldimage and luminescence image of nanoKAZ from the stable expressed cellline, pcDNA3-GLsp-nanoKAZ/CHO-K1, respectively, and labeled c and drepresent the bright-field image and luminescence image of nanoKAZ fromthe stable expressed cell line pcDNA3-nanoKAZ/CHO-K1, respectively.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below in detail.

1. Luciferase Mutants of the Invention

The term luciferase mutant of the present invention refers to a mutantof the protein with a molecular weight of 19 kDa of Oplophorusluciferase. Specifically, the luciferase mutant of the present inventionis intended to mean a luciferase mutant having substantially the sameactivity as the luciferase mutant comprising an amino acid sequence inwhich at least one amino acid selected from amino acids at the positionsof 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2.

The term substantially the same activity is intended to mean at leastone activity selected from luciferase activity, activity forextracellular secretion when expressed in animal cells in the absence ofany secretory signal peptide sequences, and so on.

The term “luciferase activity” is intended to mean the activity forcatalyzing the luminescence reaction using a luciferin (e.g.,coelenterazines) which serves as a substrate, namely, the reaction inwhich luciferin (e.g., coelenterazines) is oxidized with molecularoxygen to produce oxyluciferin in its excited state. The excited stateof oxyluciferin produced emits visible light and converts to the groundstate of oxyluciferin.

Luminescence activity can be determined by the method described in,e.g., Inouye, S. & Shimomura, O. (1977) Biochem. Biophys. Res. Commun.233, 349-353. Specifically, the luciferase mutant of the presentinvention is mixed with a luciferin to start the luminescence reaction,and the activity of catalyzing luminescence reaction can be determinedusing a luminometer. Commercially available luminometers, e.g.,Luminescencer-PSN AB2200 (manufactured by Atto Corp.) or Centro 960luminometer (manufactured by Berthold Inc.) may be used as luminometers.

The luciferin used in the present invention may be any luciferin as faras it serves as a substrate for the luciferase mutants of the presentinvention. Specifically, the luciferin used in the present inventionincludes coelenterazines containing the imidazopyrazinone ring as thebackbone.

The term coelenterazines are used to mean coelenterazine (also referredto as “native coelenterazine”) or its analogues. Coelenterazineanalogues include, for example, bis-coelenterazine, 6hf-coelenterazine,deoxyfuran-coelenterazine (furimazine), h-coelenterazine,hcp-coelenterazine, cp-coelenterazine, f-coelenterazine,fcp-coelenterazine, n-coelenterazine, MeO-coelenterazine,e-coelenterazine, cl-coelenterazine, ch-coelenterazine,3iso-coelenterazine, 3meo-coelenterazine, cf3-coelenterazine,i-coelenterazine, et-coelenterazine, me-coelenterazine,3me-coelenterazine, αmeh-coelenterazine, 8-(1-naphthyl)-coelenterazine,8-(2-naphthyl)-coelenterazine, 8-(2-thienyl)-coelenterazine,6,8-di-(2-thienyl)-coelenterazine, 8-(4-hydroxyphenyl)-coelenterazine,8-(2-benzothienyl)-coelenterazine, 8-(b-styryl)-coelenterazine,8-phenyl-coelenterazine, 6-deoxy-coelenterazine,8-(3-thienyl)-coelenterazine, and 8-(3-benzo[b]thienyl)-coelenterazine.Of these coelenterazines, bis-coelenterazine or 6h-f-coelenterazine isparticularly preferred in some embodiments of the present invention. Insome other embodiments of the present invention, h-coelenterazine orf-coelenterazine is particularly preferred.

These coelenterazines could be synthesized by publicly known methods ormay also be commercially available.

The coelenterazines could be synthesized by the methods described in,e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura et al.(1989) Biochem. J. 261, 913-920, Shimomura et al. (1990) Biochem. J.270, 309-312, Tetrahedron Lett. 38: 6405-6406, WO 2010/090319, Inouye etal. (2010) Anal. Biochem. 407, 247-252 or Inouye et al. (2013) Biocchem.Biophys. Res. Commun. 437, 23-28, or respective modifications thereof.Furimazine may be produced by the method described in Hall et al. (2012)ACS Chem. Biol. 16; 848-1857.

The coelenterazines which are commercially available include, forexample, coelenterazine, cf3-coelenterazine and h-coelenterazinemanufactured by JNC Corp.; hcp-coelenterazine, cp-coelenterazine,f-coelenterazine, fcp-coelenterazine and n-coelenterazine manufacturedby Biotium Inc.; and bis-coelenterazine manufactured by Prolume Ltd. andcoelenterazine, furimazine and h-coelenterazine manufactured by PromegaCorp.

The “luminescence activity using a luciferin as a substrate” refers toluminescence activity using preferably coelenterazines as a substrate.In a preferred embodiment of the invention, the “luminescence activityusing coelenterazines as a substrate” is the luminescence activity inwhich bis-coelenterazine or 6h-f-coelenterazine serves as the substrate.In another preferred embodiment of the invention, the “luminescenceactivity using coelenterazines as a substrate” is the luminescenceactivity in which h-coelenterazine or f-coelenterazine serves as thesubstrate.

The “activity for extracellular secretion (secreting extracellularly)when expressed in animal cells in the absence of any secretory signalpeptide sequences” is intended to mean that when the protein isexpressed in animal cells, the expressed protein is secretedextracellularly from the endoplasmic reticulum but not via thetrans-Golgi network, despite having no secretory signal peptide. The“extracellular secretion (secreting extracellularly)” refersspecifically to extracellular secretion of the protein in an amount (byweight) of 5% or more, 10% or more, or 20% or more of the expressedprotein. Specific examples of the “animal cells” are those laterdescribed. The “secretory signal peptide” is intended to mean a signalfor secretion, excluding the region in which the secretion informationof nanoKAZ is carried, specifically excluding the region of amino acidsat the positions of 1 to 4 from the amino terminus.

The “luciferase mutant having substantially the same activity as theluciferase mutant comprising an amino acid sequence in which at leastone amino acid selected from amino acids at the positions of 1 to 4 isdeleted in the amino acid sequence of SEQ ID NO: 2” includes, forexample, a luciferase mutant selected from (a) to (d) described below.

(a) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from the 1st to 4th amino acids is deletedin the amino acid sequence of SEQ ID NO: 2;

(b) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 is anamino acid sequence in which 1 to 17 amino acids are deleted,substituted, inserted and/or added in the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;

(c) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 hasat least 90% identity to the amino acid sequence at the positions of 5to 169 of SEQ ID NO: 2, and having a luciferase activity; and,

(d) a luciferase mutant comprising an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 isencoded by a polynucleotide which hybridizes under stringent conditionsto a polynucleotide consisting of a nucleotide sequence complementary toa nucleotide sequence encoding the amino acid sequence at the positionsof 5 to 169 of SEQ ID NO: 2, and having a luciferase activity.

In (a) to (d) described above, the term “at least one amino acidselected from amino acids at the positions of 1 to 4 is deleted in theamino acid sequence of SEQ ID NO: 2” is intended to mean a deletion ofat least one amino acid residue at the positions of 1 to 4 in the aminoacid sequence of SEQ ID NO: 2. Preferably, the amino acid at theposition of 1, all amino acids at the positions of 1 and 2, all aminoacids at the positions of 1 to 3 or all amino acids at the positions of1 to 4 are deleted.

Specifically, “at least one” in “at least one amino acid is deleted”refers to 1, 2, 3 or 4, preferably 1 or 2, and more preferably 1.

In (b) to (d) described above, the “amino acid sequence excluding theamino acids at the positions of 1 to 4” is intended to mean an aminoacid sequence corresponding to the amino acid sequence at the positionsof 5 to 169 of SEQ ID NO: 2 prior to the mutation, in each of the aminoacid sequences in the luciferase mutants defined in (b) to (d) describedabove.

In (b) above, the term “1 to 17 amino acids are deleted, substituted,inserted and/or added” is intended to mean that the deletion,substitution, insertion and/or addition of 1 to 17 amino acid residuesoccur at an optional position(s) in the same sequence and at 1 to 17positions in the amino acid sequence.

The range of “1 to 17” in the “1 to 17 amino acids are deleted,substituted, inserted and/or added” described above is, for example, 1to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1to 2 and 1. In general, the less the number of amino acids deleted,substituted, inserted and/or added, the more preferred. Such proteinsmay be produced by site-directed mutagenesis described in J. Sambrook,E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratorymanual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(2012); Ausbel F. M. et al., Current Protocols in Molecular Biology,Supplement 1˜38, John Wiley and Sons (1987-1997); Nuc. Acids. Res., 10,6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315(1985); Nuc. Acids. Res., 13, 4431 (1985); Proc. Natl. Acad. Sci. USA,82, 488 (1985), etc.

The position(s) of the amino acid(s) which is/are substituted in theamino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2 are notparticularly limited so long as it is other than at the positions of 11,18, 27, 33, 43, 68, 72, 75, 90, 115, 124 and 166, and include one ormore position(s) selected from the group consisting of the positions of13, 14, 15, 25, 30, 36, 70, 83, 106, 128, 153, 156, 157, 159, 162, 163and 169. In particular, the substitution positions can be at least (oneor more) position(s) selected from the group consisting of the positionsof 13, 14, 153, 159, 163 and 169.

Examples of amino acid residues which are mutually substitutable aregiven below. Amino acid residues in the same group are mutuallysubstitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine,t-butylalanine and cyclohexylalanine;

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid and 2-aminosuberic acid;

Group C: asparagine and glutamine;

Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionic acid;

Group E: proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: serine, threonine and homoserine; and,

Group G: phenylalanine and tyrosine.

In (c) described above, the range of “at least 90%” in the “amino acidsequence having at least 90% identity” is, for example, 90% or more, 91%or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% ormore, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7%or more, 99.8% or more, or 99.9% or more. In general, the numericalvalue of the identity described above is more preferable as the numberbecomes larger. The identity of amino acid sequences or nucleotidesequences can be determined using sequencing programs such as BLAST(see, e.g., Altzchul, S. F. et al., J. Mol. Biol., 215, 403 (1990)).When BLAST is used, the default parameters for the respective programsare employed.

In (d) described above, the “polynucleotide which hybridizes understringent conditions” is intended to mean a polynucleotide (e.g., DNA)which is obtained by, for example, colony hybridization, plaquehybridization or Southern hybridization using as a probe all or part ofthe polynucleotide consisting of a nucleotide sequence complementary tothe nucleotide sequence encoding the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2. Specific examples includepolynucleotides which can be identified by performing hybridization at65° C. in the presence of 0.7 to 1.0 mol/L NaCl using a filter on whichthe polynucleotide from a colony or plaque is immobilized, then washingthe filter at 65° C. with an SSC (saline-sodium citrate) solution havinga concentration of 0.1 to 2 times (1×SSC solution is composed of 150mmol/L sodium chloride and 15 mmol/L sodium citrate).

Hybridization may be performed in accordance with modifications of themethods described in laboratory manuals, e.g., J. Sambrook, E. F.Fritsch & T. Maniatis (Ed.), Molecular Cloning, A Laboratory Manual (4thedition), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012),Ausbel F. M. et al., Current Protocols in Molecular Biology, Supplement1-38, John Wiley and Sons (1987-1997), Glover D. M. and Hames B. D., DNACloning 1: Core Techniques, A practical Approach, Second Edition, OxfordUniversity Press (1995), etc.

The “stringent conditions” may be any of low stringent conditions,moderate stringent conditions or high stringent conditions. The“low-stringent conditions” are, for example, conditions of 5×SSC,5×Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 32° C. The“moderate stringent conditions” are, for example, conditions of 5×SSC,5×Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 42° C. The“high-stringent conditions” are, for example, 5×SSC, 5×Denhart'ssolution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50° C. The morestringent the conditions are, the higher the complementarity requiredfor double-strand formation. Specifically, under these conditions, forexample, a polynucleotide (e.g., DNA) of higher homology is expected tobe obtained efficiently as the temperature becomes higher, althoughmultiple factors are involved in hybridization stringency, includingtemperature, probe concentration, probe length, ionic strength, time,base concentration, etc. One skilled in the art may achieve a similarstringency by appropriately choosing these factors.

When commercially available kits are used for the hybridization, forexample, Alkphos Direct Labeling Reagents (manufactured by GE HealthcareInc.) can be used. In this case, according to the protocol attached, amembrane is incubated with a labeled probe overnight, the membrane iswashed with a primary wash buffer containing 0.1% (w/v) SDS underconditions at 55° C. and then the hybridized DNA can be detected.

Other hybridizable polynucleotides include, as calculated by asequencing program such as BLAST or the like using the defaultparameters, DNAs having the identity of approximately 60% or more, 65%or more, 70% or more, 75% or more, 80% or more, 85% or more, 88% ormore, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more,99% or more, 99.3% or more, 99.5% or more, 99.7% or more, 99.8% or more,or 99.9% or more, to the polynucleotide consisting of the nucleotidesequence of SEQ ID NO: 2. The identity of nucleotide sequences can bedetermined using the method described above.

In a preferred embodiment of the invention, the luciferase mutant is aluciferase mutant comprising the amino acid sequence of SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, more preferably, aluciferase mutant comprising the amino acid sequence of SEQ ID NO: 4,SEQ ID NO: 6 or SEQ ID NO: 8, and most preferably, a luciferase mutantcomprising the amino acid sequence of SEQ ID NO: 4.

The luciferase mutant of the present invention may further contain anadditional peptide sequence at the N terminus and/or C terminus,preferably at the N terminus. The additional peptide sequence is atleast one peptide sequence selected from the group consisting of apeptide sequence for purification, a peptide sequence for expressing theluciferase mutant of the present invention as a soluble protein and anepitope sequence capable of recognizing an antibody. The additionalpeptide sequence is preferably a peptide sequence for purification. Inanother preferred embodiment of the invention, the additional peptidesequence is at least one sequence selected from the group consisting ofa peptide sequence for purification and a peptide sequence forexpressing the luciferase mutant of the present invention as a solubleprotein.

Peptide sequences employed in the art may be used as the peptidesequence for purification. The peptide sequence for purificationincludes, for example, a histidine tag sequence with a consecutive aminoacid sequence of at least 4 histidine residues and preferably at least 6residues, an amino acid sequence with a binding domain of glutathioneS-transferase into glutathione, the amino acid sequence of Protein A,etc.

The peptide used to express the luciferase mutant of the presentinvention as a soluble protein includes, for example, polypeptidesrepresented by formula (Z)_(n). The amino acid sequences for thepolypeptides represented by formula (Z)_(n) and the nucleic acidsequences encoding the same are described in, e.g., JPA KOKAI No.2008-99669.

Peptide sequences used in the art can be used as the epitope sequencecapable of recognizing an antibody.

In some embodiments of the present invention, the additional peptidesequence in the luciferase mutant does not carry a secretory signalpeptide sequence. The “secretory signal peptide sequence” includes asecretory peptide sequence of Gaussia luciferase, or the like.

The method for acquiring the luciferase mutant of the invention is notparticularly limited. The luciferase mutant of the invention may be aprotein synthesized by chemical synthesis, or a recombinant proteinproduced by a genetic engineering technique. When the luciferase mutantof the invention is to be chemically synthesized, synthesis may becarried out by, for example, the Fmoc (fluorenylmethyloxycarbonyl)method or the tBoc (t-butyloxycarbonyl) method. In addition, peptidesynthesizers available from Advanced ChemTech, PerkinElmer, Pharmacia,Protein Technology Instrument, Synthecell-Vega, PerSeptive, ShimadzuCorporation, etc. may also be used for chemical synthesis. When theluciferase mutant of the invention is to be produced by a geneticengineering technique, the mutant may be produced by a conventionalgenetic recombination technique. More specifically, the luciferasemutant of the invention may be produced by inserting a polynucleotide(e.g., a DNA) encoding the luciferase mutant of the invention into asuitable expression system. The polynucleotide encoding the luciferasemutant of the invention, expression of the luciferase mutant of theinvention in an expression system or the like will be later described.

2. Polynucleotide of the Invention

The present invention also provides a polynucleotide comprising apolynucleotide encoding the luciferase mutant of the invention describedabove. The polynucleotide of the invention may be any polynucleotide solong as it has a nucleotide sequence encoding the luciferase mutant ofthe invention, although a DNA is preferred. Examples of the DNA includegenomic DNA, genomic DNA library, cellular or tissue cDNA, cellular ortissue cDNA library, synthetic DNA, etc. Vectors used in the librariesare not particularly limited and may be any of bacteriophages, plasmids,cosmids, phagemids, etc. Also, these vectors may be amplified directlyby a Reverse Transcription Polymerase Chain Reaction (hereinafterabbreviated as RT-PCR) using the total RNA or mRNA fraction preparedfrom the cell or tissue described above.

The polynucleotide of the invention includes the followingpolynucleotides (i) to (iv).

(i) A polynucleotide comprising a polynucleotide encoding the luciferasemutant described in (a) above;

(ii) A polynucleotide comprising a polynucleotide encoding theluciferase mutant described in (b) above;

(iii) A polynucleotide comprising a polynucleotide encoding theluciferase mutant described in (c) above; and,

(iv) A polynucleotide comprising a polynucleotide encoding theluciferase mutant described in (d) above.

A polynucleotide encoding a protein having a given amino acid sequence,in which one or more amino acids are substituted in the amino acidsequence, can be obtained by using a site-specific mutagenesis technique(see, e.g., Gotoh, T. et al., Gene 152, 271-275 (1995), Zoller, M. J.,and Smith, M., Methods Enzymol. 100, 468-500 (1983), Kramer, W. et al.,Nucleic Acids Res. 12, 9441-9456 (1984), Kramer W, and Fritz H. J.,Methods. Enzymol. 154, 350-367 (1987), Kunkel, T. A., Proc. Natl. Acad.Sci. USA. 82, 488-492 (1985), Kunkel, Methods Enzymol. 85, 2763-2766(1988); etc.), the methods using amber mutation (see, e.g., the gappedduplex method, Nucleic Acids Res., 12, 9441-9456 (1984), etc.), etc.

Alternatively, mutations may also be introduced into the polynucleotideby PCR (cf, e.g., Ho S. N. et al., Gene, 77, 51 (1989), etc.) using apair of primers bearing on the respective 5′ ends a sequence in whichthe targeted mutation (deletion, addition, substitution and/orinsertion) has been introduced.

Also, a polynucleotide encoding a partial fragment of protein, which isone type of the deletion mutant, can be obtained using as the primers anoligonucleotide having a sequence which matches the nucleotide sequenceat the 5′ end of the region encoding the partial fragment to be producedin the polynucleotide encoding the target protein and an oligonucleotidehaving a sequence complementary to the nucleotide sequence at the 3′ endthereof, and performing PCR in which the polynucleotide encoding thetarget protein is used as a template.

The polynucleotide of the present invention includes preferably apolynucleotide comprising a polynucleotide encoding the luciferasemutant comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8 or SEQ ID NO: 10, more preferably, a polynucleotidecomprising a polynucleotide encoding the luciferase mutant comprisingthe amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8,and most preferably, a polynucleotide comprising a polynucleotideencoding the luciferase mutant comprising the amino acid sequence of SEQID NO: 4.

The polynucleotide encoding the luciferase mutant comprising the aminoacid sequence of SEQ ID NO: 2 includes a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 1. The polynucleotide encoding theluciferase mutant comprising the amino acid sequence of SEQ ID NO: 4includes a polynucleotide comprising the nucleotide sequence of SEQ IDNO: 3. The polynucleotide encoding the luciferase mutant comprising theamino acid sequence of SEQ ID NO: 6 includes a polynucleotide comprisingthe nucleotide sequence of SEQ ID NO: 5. The polynucleotide encoding theluciferase mutant comprising the amino acid sequence of SEQ ID NO: 8includes a polynucleotide comprising the nucleotide sequence of SEQ IDNO: 7. The polynucleotide encoding the luciferase mutant comprising theamino acid sequence of SEQ ID NO: 10 includes a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 9.

In some embodiments of the present invention, the polynucleotide ispreferably a polynucleotide comprising a polynucleotide consisting ofthe nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 orSEQ ID NO: 9, more preferably, a polynucleotide comprising apolynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3,SEQ ID NO: 5 or SEQ ID NO: 7, and most preferably, a polynucleotidecomprising a polynucleotide consisting of the nucleotide sequence of SEQID NO: 3.

The polynucleotide of the present invention may further contain apolynucleotide encoding an additional peptide sequence at the 5′ endand/or 3′ end, preferably at the 5′ end. The polynucleotide encoding theadditional peptide sequence includes a polynucleotide encoding at leastone peptide sequence selected from the group consisting of a peptidesequence for purification, a peptide sequence for expressing theluciferase mutant of the present invention as a soluble protein, anepitope sequence capable of recognizing an antibody, and the like.

Polynucleotides comprising nucleotide sequences encoding the peptidesequence for purification employed in the art can be used as thepolynucleotide encoding the peptide sequence for purification. Examplesof the peptide sequence for purification include those as describedabove.

The polynucleotide encoding the peptide sequence used to express theluciferase mutant of the present invention as a soluble proteinincludes, for example, polypeptides represented by formula (Z)_(n). Theamino acid sequences for the polypeptides represented by formula (Z)_(n)and the nucleic acid sequences encoding the same are those as describedabove.

Polynucleotides comprising nucleotide sequences encoding the epitopesequence capable of recognizing antibodies which are used in the art canbe used as the polynucleotide encoding the antibody-recognizing epitopesequence.

In some embodiments of the present invention, a polynucleotide as thepolynucleotide encoding the additional peptide sequence does not includepolynucleotides encoding a secretory signal peptide sequence. The“secretory signal peptide sequence” includes those as described above,and the like.

3. Recombinant Vector and Transformant of the Invention

The present invention further provides recombinant vectors andtransformants comprising the polynucleotides of the present inventiondescribed above.

Preparation of Recombinant Vector

The recombinant vector of the invention can be obtained by ligating(inserting) the polynucleotide (DNA) of the invention to (into) anappropriate vector. More specifically, the recombinant vector can beobtained by digesting the purified polynucleotide (DNA) with a suitablerestriction enzyme, then inserting into a suitable vector at therestriction enzyme site or multiple-cloning site, and ligating to thevector. The vector for inserting the polynucleotide of the invention isnot particularly limited as long as it is replicable in a host, andincludes plasmids, bacteriophages, animal viruses, etc. Examples ofplasmids include plasmids from E. coli (e.g., pBR322, pBR325, pUC118,pUC119, etc.), plasmids from Bacillus subtilis (e.g., pUB110, pTP5,etc.) and plasmids from yeast (e.g., YEp13, YEp24, YCp50, etc.).Examples of bacteriophages include, e.g., λ phage. Examples of animalviruses include retroviruses, vaccinia viruses and insect viruses (e.g.,baculoviruses). In addition, a pCold I vector, a pCold II vector, apCold III vector and a pCold IV vector (all manufactured by Takara BioInc.), a pcDNA3 vector, a PICZa vector (manufactured by Invitrogen Inc.)and the like may also be suitably used.

The polynucleotide of the present invention is generally ligated in anexpressible manner downstream of a promoter in a suitable vector. Whenthe host used for transformation is an animal cell, the promoter ispreferably an SV40-derived promoter, retrovirus promoter,metallothionein promoter, heat shock promoter, cytomegalovirus promoter,SRα promoter, and so on. When the host is a bacterium of the genusEscherichia, Trp promoter, T7 promoter, lac promoter, recA promoter, λPLpromoter, lpp promoter, etc. are preferred. When the host is a bacteriumof the genus Bacillus, SPO1 promoter, SPO2 promoter, penP promoter, etc.are preferred. When the host is yeast, PHO5 promoter, PGK promoter, GAPpromoter, ADH1 promoter, GAL promoter, etc. are preferred. When the hostis an insect cell, polyhedrin promoter, P10 promoter, etc. arepreferred.

A low-temperature expression-inducible promoter may also be suitablyused. Examples of the low-temperature expression-inducible promoterinclude promoter sequences for cold shock genes. The cold shock geneincludes, for example, E. coli cold shock genes (e.g., cspA, cspB, cspG,cspI and csdA), Bacillus caldolyticus cold shock genes (e.g., Bc-Csp),Salmonella enterica cold shock genes (e.g., cspE) and Erwinia carotovoracold shock genes (e.g., cspG). Among others, cspA promoter, cspBpromoter, cspG promoter, cspI promoter, csdA promoter and the like canbe advantageously used as the low-temperature expression-induciblepromoter.

In addition to the foregoing, the recombinant vector of the inventionmay further contain, if desired, an enhancer, a splicing signal, a polyAaddition signal, a ribosome binding sequence (SD sequence), a selectionmarker, etc., and provided for use. The selection marker includes, forexample, a dihydrofolate reductase gene, an ampicillin resistance gene,a neomycin resistance gene, etc.

Preparation of Transformant

The thus obtained recombinant vector comprising the polynucleotide ofthe invention is introduced into an appropriate host to prepare thetransformant. The host is not particularly limited as long as it iscapable of expressing the polynucleotide (DNA) of the invention, and maybe bacteria of the genera Escherichia, Bacillus, Pseudomonas andRhizobium, yeast, animal cells or insect cells, etc. Bacteria of thegenus Escherichia include Escherichia coli, etc. Bacteria of the genusBacillus include Bacillus subtilis, etc. Bacteria of the genusPseudomonas include Pseudomonas putida, etc. Bacteria of the genusRhizobium include Rhizobium meliloti, etc. Yeast includes Saccharomycescerevisiae, Schizosaccharomyces pombe, etc. Animal cells include primarycell cultures, iPS cells, cultured cell lines (CHO cells, HEK293 cells,HL-60 cells, HeLa cells, MDCK cells, NIH3T3cells, PC12 cells), etc.Insect cells include Sf9, Sf21, etc.

The method of transfecting the recombinant vector into the host and themethod of transformation by the same can be performed according tovarious general methods. The method for transfecting the recombinantvector into the host cell includes the calcium phosphate method(Virology, 52, 456-457 (1973)), the lipofection method (Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)), the electroporation method (EMBO J.,1, 841-845 (1982)), etc. The method for transformation of the bacteriaof the genus Escherichia includes the methods described in, e.g., Proc.Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), etc. Themethod for transformation of the bacteria of the genus Bacillus includesthe method described in Molecular & General Genetics, 168, 111 (1979),etc. The method for transforming yeast includes the method described inProc. Natl. Acad. Sci. USA, 75, 1929 (1978), etc. The method fortransformation of animal cells includes the method described inVirology, 52, 456 (1973), etc. The method for transformation of insectcells includes the method described in Bio/Technology, 6, 47-55 (1988),etc. Thus, the transformant transformed with the recombinant vectorcomprising the polynucleotide encoding the luciferase mutant of theinvention (the polynucleotide of the invention) can be obtained.

Expression Vector and Transformant Comprising Low-TemperatureExpression-Inducible Promotor Sequence

An expression vector comprising the low-temperature expression-induciblepromoter sequence is preferred as the expression vector among others.

Specifically, the expression vector comprising the low-temperatureexpression-inducible promoter sequence is intended to mean an expressionvector comprising the following promoter sequence and coding sequence:

(1) a low-temperature expression-inducible promoter sequence; and,

(2) a coding sequence comprising the polynucleotide of the invention.

The low-temperature expression-inducible promoter sequence is intendedto mean a promoter sequence which is capable of inducing expression ofthe protein by lowering the temperature from the culture conditionsunder which host cells can grow. Examples of the low-temperatureexpression-inducible promoter are promoters for genes encoding coldshock proteins (cold shock genes). Examples of the cold shock genepromoters include those as described above.

The temperature at which the low-temperature expression-induciblepromoter used in the invention is capable of inducing expression isgenerally 30° C. or less, preferably 25° C. or less, more preferably 20°C. or less, and most preferably 15° C. or less. In order to induce theexpression more efficiently, however, the expression induction isgenerally performed at 5° C. or more, preferably at 10° C. or more, andmost preferably at approximately 15° C.

In preparing the expression vector of the invention comprising thelow-temperature expression-inducible promoter sequence, the pCold Ivector, pCold II vector, pCold III vector and pCold IV vector (allmanufactured by Takara Bio Inc.) can be suitably used as the vector forinsertion of the polynucleotide of the invention. The protein can beproduced as a soluble protein in the cytoplasm in a host cell whenexpression is performed in a prokaryotic host cell using these vectors.

Prokaryotic cells are preferred as the host into which the expressionvector comprising the low-temperature expression-inducible promotersequence is introduced, more preferably, E. coli, and particularlypreferably, the BL21 and JM109 strains. Among others, the BL21 strain ismost preferred. The BL21 and JM109 strains are available from, e.g.,Novagen.

Temperatures for incubation at which the transformant carrying theexpression vector comprising the low-temperature expression-induciblepromoter sequence grows are generally 25 to 40° C. and preferably 30 to37° C. Temperatures for inducing the expression are generally 4 to 25°C., preferably 10 to 20° C., more preferably 12 to 18° C., and mostpreferably 15° C.

4. Production of Luciferase Mutant of the Invention

The present invention further provides a method for producing theluciferase mutant of the invention, which comprises the steps ofculturing the transformant described above to produce the luciferasemutant of the invention. The luciferase mutant of the invention can beproduced, for example, by culturing the transformant described aboveunder conditions where the polynucleotide (DNA) encoding the luciferasemutant of the invention can be expressed, producing/accumulating andthen separating/purifying the luciferase mutant of the invention.

Incubation of Transformant

The transformant of the invention can be incubated in a conventionalmanner used for incubation of a host. By the incubation, the luciferasemutant of the invention is produced by the transformant and accumulatedwithin the transformant or in the culture medium.

The medium used for culturing the transformant using bacteria of thegenus Escherichia or the genus Bacillus as a host may be any of anatural medium and a synthetic medium as far as it is a medium whichcontains carbon sources, nitrogen sources, inorganic salts, etc.necessary for growth of the transformant, and in which the transformantcan efficiently grow. Examples of carbon sources which can be used arecarbohydrates such as glucose, fructose, sucrose, starch, etc.; organicacids such as acetic acid, propionic acid, etc.; alcohols such asethanol, propanol, and the like. Examples of nitrogen sources which canbe used include ammonia, ammonium salts of inorganic or organic acidssuch as ammonium chloride, ammonium sulfate, ammonium acetate, ammoniumphosphate, etc., and other nitrogen-containing compounds, and furtherinclude peptone, meat extracts, corn steep liquor, and the like.Examples of inorganic salts include monobasic potassium phosphate,dibasic potassium phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate,calcium carbonate, etc. If necessary, antibiotics such as ampicillin ortetracycline can be added to the medium during incubation. Where thetransformant transformed by the expression vector using an induciblepromoter as the promoter is cultured, an inducer may also be added tothe medium, if necessary. For example, when the transformant transformedby an expression vector using a Lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside (IPTG), etc. may be added to themedium and indoleacrylic acid (IAA), etc. may be added to the medium inculturing the transformant transformed by an expression vector using atrp promoter.

When the host is bacteria of the genus Escherichia, incubation isperformed generally at approximately 15 to 43° C. for approximately 3 to24 hours. If necessary, aeration and agitation may be applied. When thehost is bacteria of the genus Bacillus, incubation is performedgenerally at approximately 30 to 40° C. for approximately 6 to 24 hours.If necessary, aeration and agitation may be applied.

Media for incubation of the transformant when the host is yeast includeBurkholder's minimal medium (Proc. Natl. Acad. Sci. USA, 77, 4505(1980)) and an SD medium containing 0.5% (w/v) Casamino acids (Proc.Natl. Acad. Sci. USA, 81, 5330 (1984)). Preferably, the pH of the mediumis adjusted to approximately 5 to 8. Incubation is performed generallyat approximately 20 to 35° C. for approximately 24 to 72 hours. Ifnecessary, aeration and agitation may be applied.

Media for culturing the transformant when the host is an animal cellinclude MEM medium supplemented with approximately 5 to 20% (v/v) fetalcalf serum (Science, 122, 501 (1952)), DMEM medium (Virology, 8, 396(1959)), etc. Preferably, the pH of the medium is adjusted toapproximately 6 to 8. Incubation is performed generally at approximately30 to 40° C. for approximately 15 to 60 hours. If necessary, aerationand agitation may be applied.

Media for culturing the transformant when the host is an insect cellinclude Grace's insect medium (Nature, 195, 788 (1962)) to whichadditives such as 10% (v/v) immobilized bovine serum are suitably added.Preferably, the pH of the medium is adjusted to approximately 6.2 to6.4. Incubation is performed generally at approximately 27° C. forapproximately 3 to 5 days. If necessary, aeration and agitation may beapplied.

Temperatures for incubation at which the transformant transformed by theexpression vector comprising the low-temperature expression-induciblepromoter sequence and temperatures for expression induction are asdescribed above.

Separation/Purification of Luciferase Mutant of the Invention

The luciferase mutant of the invention can be obtained byseparating/purifying the luciferase mutant of the invention from theculture described above. As used herein, the culture is intended to meanany one of a culture broth, cultured cells or cultured bacteria and acell lysate of the cultured cells or cultured bacteria. The luciferasemutant of the invention can be separated/purified in a conventionalmanner.

Specifically, when the luciferase mutant of the invention accumulates inthe cultured bacteria or cultured cells, after completion of theincubation the bacteria or cells are disrupted in a conventional manner(e.g., ultrasonication, lysozyme, freezing and thawing, etc.), and thena crude extract of the luciferase mutant of the invention can beobtained in a conventional manner (e.g., centrifugation, filtration,etc.). When the luciferase mutant of the invention accumulates in theperiplasmic space, after completion of the incubation the extractcontaining the luciferase mutant of the invention can be obtained in aconventional manner (e.g., the osmotic shock method, etc.). When theluciferase mutant of the invention accumulates in the culture broth,after completion of the incubation the culture supernatant containingthe luciferase mutant of the invention can be obtained by separating thebacteria or cells and the culture supernatant in a conventional manner(e.g., centrifugation, filtration, etc.).

The luciferase mutant of the invention contained in the extract orculture supernatant thus obtained can be purified by conventionalmethods of separation and purification. Examples of these methods forseparation and purification which may be used include ammonium sulfateprecipitation, gel filtration chromatography, ion-exchangechromatography, affinity chromatography, reversed-phase high-performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or in asuitable combination thereof. When the luciferase mutant of theinvention contains the peptide sequence for purification describedabove, it is preferred to perform purification using the same.Specifically, when the luciferase mutant of the invention contains ahistidine tag sequence, nickel chelate affinity chromatography may beused; when the luciferase mutant of the invention contains the bindingdomain of S-transferase to glutathione, affinity chromatography with aglutathione-binding gel may be used; when the luciferase mutant of theinvention contains the amino acid sequence of Protein A, antibodyaffinity chromatography may be used.

5. Use of Luciferase Mutant of the Invention

Use as Detection Marker by Luminescence

The luciferase mutant of the invention can be used as a detection markerwhich emits luminescence in the presence of a luciferin (hereinafter“detection marker of the present invention”). The detection marker ofthe present invention can be utilized for detection of the targetsubstance in, e.g., an immunoassay, a hybridization assay, etc.

The luciferase mutant of the invention can be expressed, e.g., as afusion protein with a target protein, and introduced into cells by meansof the microinjection method, etc., and the resulting product can beused to determine distribution of the target protein described above.The distribution of such a target protein or the like can be determinedby using detection methods such as luminescence imaging. In addition tothe introduction into cells by means of the microinjection method or thelike, the luciferase mutant of the invention can be expressed in cellsto provide for use.

The luminescence substrate (luciferin) used is preferablycoelenterazines, and particularly preferably, bis-coelenterazine or6h-f-coelenterazine, as described above. Bis-coelenterazine or6h-f-coelenterazine displays the luminescence activity approximately 10times higher than that of coelenterazine and emits glow.

Use as Reporter Protein

The luciferase mutant of the invention may also be used as a reporterprotein to assay the transcription activity of promoters, etc. In thiscase, the polynucleotide of the invention is used as a reporter gene andthe luciferase mutant encoded by the reporter gene is contacted withluciferin. As used herein, the term “contact” is intended to mean thatthe luciferase mutant of the invention and a luciferin are allowed to bepresent in the same reaction system or culture system, which includes,for example, addition of a luciferin to a culture container charged withcells expressing the luciferase mutant of the invention, mixing thecells with a luciferin, and incubation of the cells in the presence of aluciferin. The polynucleotide encoding the luciferase mutant of theinvention (i.e., the polynucleotide of the invention) is fused to atarget promoter or some other expression control sequence (e.g., anenhancer, etc.) to construct a vector. By introducing the vector into ahost cell and detecting the luminescence from the luciferase mutant ofthe invention in the presence of a luciferin (luminescence substrate),the activity of the target promoter or some other expression controlsequence can be assayed. Furthermore, the expressed luciferase mutant isreacted with coelenterazines and the luminescence generated may also bevisualized in pictures by using a high-sensitive detector.

The luciferin used is preferably coelenterazines, and particularlypreferably, bis-coelenterazine or 6h-f-coelenterazine, as describedabove. Bis-coelenterazine or 6h-f-coelenterazine displays theluminescence activity approximately 10 times higher than that ofcoelenterazine and emits glow.

The cells used are preferably animal cells. In the case of animal cells,the luciferase mutant in a preferred embodiment of the invention issecretion outside of cells.

The polynucleotide of the invention can be used as a reporter gene insuch a manner as described above.

Method for Visualizing Luminescence Reaction

The luciferase mutant of the present invention can be used in the methodfor visualizing luminescence activities. By “visualizing luminescenceactivities,” for example, the pattern of secretion can be observed whenthe luciferase mutant of the present invention is secretedextracellularly. The luciferase mutant of the invention is secretedextracellularly without passing through the trans-Golgi network, as willbe demonstrated in EXAMPLES described below.

Specifically, the method for visualizing a luminescence reactioncomprises contacting the luciferase mutant of the invention with aluciferin. As used herein, the term “contact” is intended to mean thatthe luciferase mutant of the invention and a luciferin are allowed to bepresent in the same reaction system or culture system, which includes,for example, addition of a luciferin to a culture container charged withcells expressing the luciferase mutant of the invention, mixing thecells with a luciferin and incubation of the cells in the presence of aluciferin. A vector comprising the polynucleotide encoding theluciferase mutant of the invention (i.e., the polynucleotide of theinvention) is constructed. By introducing the vector into a host celland detecting the luminescence from the luciferase mutant of theinvention in the presence of a luciferin (luminescence substrate), theluminescence reaction can be visualized. In this case, the luminescenceis detected by using, e.g., a high-sensitive detector.

The luciferin used is preferably coelenterazines, and particularlypreferably, h-coelenterazine or f-coelenterazine, as described above.H-coelenterazine or f-coelenterazine displays the luminescence activity10 times higher than that of coelenterazine and its light decays fast.

The cells used are preferably animal cells. Even in animal cells, theluciferase mutant in a preferred embodiment of the invention is secretedextracellularly.

The polynucleotide of the invention can be used in the method forvisualizing a luminescence reaction in such a manner as described above.

Material for Amusement Supplies

The luciferase mutant of the invention has the activity of catalyzingthe reaction where a luciferin is oxidized with oxygen molecules to formoxyluciferin in its excited state. The oxyluciferin in the excited stateemits visible light to decay to the ground state. Accordingly, theluciferase mutant of the invention can be used preferably as aluminescent material for amusement supplies. Examples of such amusementsupplies are luminescent soap bubbles, luminescent ice bars, luminescentcandies, luminescent color paints, etc. These amusement supplies of theinvention can be prepared in a conventional manner.

The luciferin used is preferably coelenterazines, and particularlypreferably, bis-coelenterazine or 6h-f-coelenterazine, as describedabove. Bis-coelenterazine or 6h-f-coelenterazine displays theluminescence activity approximately 10 times higher than that ofcoelenterazine and emits glow.

Bioluminescence Resonance Enemy Transfer (BRET) Method

By utilizing the principle of interaction between molecules by thebioluminescence resonance energy transfer (BRET) method, the luciferasemutant of the invention is available for analytical methods such asanalysis of physiological functions, assay of enzyme activities, etc.

For instance, when the luciferase mutant of the invention of theinvention is used as a donor and the fluorescent substance (e.g., anorganic compound, a fluorescent protein, etc.) is used as an acceptor,the interactions between the donor and acceptor above can be detected byinducing bioluminescence resonance energy transfer (BRET) between them.

In an embodiment of the present invention, the organic compound used asan acceptor includes Hoechist3342, Indo-1, DAP1, etc. In anotherembodiment of the present invention, the fluorescent protein used as anacceptor includes a green fluorescent protein (GFP), a blue fluorescentprotein (BFP), a muted GFP fluorescent protein, phycobilin, etc. Theseorganic compounds and fluorescent proteins are commercially available.

In a preferred embodiment of the present invention, the physiologicalfunctions to be analyzed include an orphan receptor (especially, a Gprotein-coupled receptor), apoptosis, transcription regulation by geneexpression, etc. In a further preferred embodiment of the presentinvention, the enzyme to be analyzed is protease, esterase, kinase, orthe like.

Analysis of the physiological functions by the BRET method can beperformed by known methods, for example, by modifications of the methoddescribed in Biochem. J. 2005, 385, 625-637 or Expert Opin. Ther Tarets,2007 11: 541-556. Enzyme activities may also be assayed by knownmethods, for example, by modifications of the method described in NatureMethods 2006, 3:165-174 or Biotechnol. J. 2008, 3:311-324.

The luminescence substrate (luciferin) used is preferablycoelenterazines, and particularly preferably, bis-coelenterazine or6h-f-coelenterazine, as described above. Bis-coelenterazine or6h-f-coelenterazine displays the luminescence activity approximately 10times higher than that of coelenterazine and emits glow.

6. Kit of the Invention

The present invention also provides a kit comprising any one selectedfrom the luciferase mutant of the invention, the polynucleotide of theinvention, the recombinant vector of the invention and the transformantof the invention. The kit of the invention may further contain aluciferin.

The luciferin is preferably coelenterazines, as described above. In someembodiments of the present invention, bis-coelenterazine or6h-f-coelenterazine is particularly preferred. In some other embodimentsof the present invention, h-coelenterazine or f-coelenterazine isparticularly preferred in some embodiments of the present invention.

The kit of the present invention may be prepared with conventionalmaterials by conventional methods. The kit of the present invention mayfurther contain, e.g., sample tubes, plates, instructions for the kituser, solutions, buffers, reagents, and samples suitable forstandardization or control samples. The kit of the present invention mayfurther contain salts including halide ions.

The kit of the present invention can be used for the aforesaidmeasurement using a reporter protein or a reporter gene, the detectionmarker with luminescence, or the analysis of physiological functions ormeasurement of enzyme activities by the BRET method. The kit can also beused for the method for luminescence reaction as described below.

7. Method for Luminescence Reaction

Luminescence Activity

The luciferase mutant of the invention has the ability of catalyzing thereaction which involves oxidization of a luciferin with oxygen moleculesto form an oxyluciferin in its excited state. The oxyluciferin in theexcited state emits light on returning to the ground state. That is, theluciferase mutant of the invention catalyzes the luminescence reactionin which a luciferin serves as a substrate to cause luminescence. Thisactivity is sometimes referred to as “the luminescence activity” in thespecification.

Luminescence Reaction

The luminescence reaction using the luciferase mutant of the inventionin which a luciferin serves as a substrate can be performed bycontacting the luciferase mutant of the invention with the luciferin. Asused herein, the term “contact” is intended to mean that the luciferasemutant of the invention and a luciferin are allowed to be present in thesame reaction system, which includes, for example, addition of theluciferase mutant of the invention to a container charged with aluciferin, addition of a luciferin to a container charged with theluciferase mutant of the invention and mixing the luciferase mutant ofthe invention with a luciferin. The reaction can be carried out underconditions conventionally used for the luminescence reaction usingOplophorus luciferase or under conditions modified therefrom.

Specifically, solvents for the reaction which are employed are, forexample, a buffer solution such as Tris-HCl buffer, sodium phosphatebuffer, etc., water, and the like.

Temperatures for the reaction are generally approximately 4° C. to 40°C. and preferably approximately 4° C. to 25° C.

In the reaction solution, pH is generally approximately 5 to 10,preferably approximately 6 to 9, more preferably approximately 7 to 8and most preferably approximately 7.5.

The luciferin is preferably coelenterazines, as described above. In someembodiments of the present invention, bis-coelenterazine or6h-f-coelenterazine is particularly preferred. In some other embodimentsof the present invention, h-coelenterazine or f-coelenterazine isparticularly preferred.

The luciferin may also be added to the reaction system in the form of asolution in a polar solvent such as dimethylformamide,dimethylsulfoxide, etc., or in an alcohol such as methanol, ethanol,butanol, etc.

Activation of Luminescence Activity

The luminescence activity of the luciferase mutant of the invention canbe activated by halide ions, nonionic surfactants, etc.

Examples of the halide ions are fluorine ions, chlorine ions, bromineions and iodine ions; preferred are chlorine ions, bromine ions andiodine ions.

The concentration of halide ions is generally approximately 10 μM to 100mM, preferably approximately 100 μM to 50 mM and particularly preferablyapproximately 1 mM to 20 mM.

The addition of the halide ions to the reaction system is performed by amethod which comprises adding them in a salt form. The salts used arealkali metal salts such as sodium salts, potassium salts, etc.; alkalineearth metal salts such as calcium salts, magnesium salts, barium salts,etc. More specific examples are NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI,CaF₂, CaCl₂, CaBr₂, CaI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, etc.

Examples of nonionic surfactants which are commercially available (tradename) include Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 80(polyoxyethylene sorbitan monooleate), Triton X-100 (polyethyleneglycol-p-isooctylphenyl ether), Briji-58 (polyoxyethylene (20) cetylether), Nonidet P-40 (ethylphenolpoly(ethylene glycol ether)n), etc.,and preferably, Tween 20, Triton X-100, etc. These surfactants arecommercially available from, e.g., Wako Pure Chemical Industries, Ltd.,Tokyo Chemical Industry Co., Ltd. and Sigma Aldrich.

Concentration of the nonionic surfactant is generally approximately0.0002% (w/v) to 0.2% (w/v), preferably, approximately 0.001% (w/v) to0.1% (w/v), and particularly preferably, approximately 0.05% (w/v) to0.02% (w/v).

Regardless of their purposes, all of the documents and publicationsdescribed in the specification are incorporated herein by reference,each in its respective entirety.

Unless otherwise indicated with respect to the embodiments and workingexamples, the methods described in standard sets of protocols such as J.Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, alaboratory manual (4th edition), Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (2012); F. M. Ausubel, R. Brent, R. E. Kingston, D. D.Moore, J. G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols inMolecular Biology, John Wiley & Sons Ltd., etc. or modifications orvariations thereof are used. When commercially available reagent kits ormeasuring apparatuses are used, protocols attached to them are usedunless otherwise indicated.

The objects, characteristics and advantages of the present invention aswell as the idea thereof are apparent to those skilled in the art fromthe descriptions given herein. Based on the description given herein,those skilled in the art can easily reproduce the present invention.

It can be understood that the embodiments of the invention, specificworking examples, etc. are disclosed as preferred embodiments of thepresent invention. These descriptions are only for illustrative andexplanatory purposes and are not intended to restrict the inventionthereto. It is further apparent to those skilled in the art that variousmodifications may be made based on the descriptions given herein withinthe intent and scope of the present invention disclosed herein.

EXAMPLES

Hereinafter, the present invention will be described with reference tospecific examples but is not deemed to be limited thereto.

Example 1 Preparation of Mutated 19kOLase Gene (dnKAZ) with 16 Mutations

Gene amplification was performed by PCR using a template ofpCold-ZZ-P-nanoKAZ described in Inouye et al (2013) Biochem. Biophys.Res. Commun. 437: 23-28 and the following primers.

Primer nanoKAZ-1N/EcoRI (SEQ ID NO: 19: 5′gcgGAATTCTTCACCCTGGAGGACTTCGTCGGC 3′: EcoRI sequence underlined)Primer nanoKAZ-3CabaI (SEQ ID NO: 20: 5′gccTCTAGATTAGGCCAGGATTCTCTCGCACAGTCT 3′: XbaI sequence underlined)

Herein, the nucleotide sequence and amino acid sequence ofpCold-ZZ-P-nanoKAZ are shown by SEQ ID NO: 11 and SEQ ID NO: 12,respectively.

Example 2 Secretory Expression Vectors for dnKAZ Using the SecretorySignal Pepetide Sequence of Gaussia Luciferase

The expression vector for dnKAZ was constructed as follows. Firstly, anovel expression vector pcDNA3-GLsp in animal cultured cells wasconstructed. Specifically, the secretory signal peptide sequence ofGaussia luciferase was obtained from pcDNA3-GLuc vector (manufactured byProlume Ltd.) by PCR using the following primers.

Primer GLsp-1R/EcoRI (SEQ ID NO: 21: 5′ggc GAA TTC GGT GGG CTT GGC CTC GGC CAC 3′, EcoRI sequence underlined)T7 primer (SEQ ID NO: 22: 5′ TAATACG ACTCACTATAGGG 3′)

After digestion with HindIII/EcoRI, the resultant fragment was insertedinto the HindIII-EcoRI site of pcDNA3 vector (manufactured by InvitrogenInc.) to construct a novel expression vector pcDNA3-GLsp. That is, thenovel expression vector is under the control of the CMV promoter,followed by the Kozak sequence, the secretory signal pepetide sequenceof Gaussia luciferase and a multiple-cloning site sequence.

Next, the expression vector for dnKAZ was constructed as follows, usingthe novel expression vector pcDNA3-GLsp. The DNA fragment obtained inEXAMPLE 1 was digested with the restriction enzymes of EcoRI/XbaI in aconventional manner and then ligated to the EcoRI-XbaI site ofpcDNA3-GLsp to construct the expression vector pcDNA3-GLsp-dnKAZ. Thegene sequence inserted was confirmed by sequencing using a DNA Sequencer(manufactured by ABI Inc.)

Example 3 Preparation of nanoKAZ Gene Fragments with the Deletion ofAmino Acid Sequences at the Amino Terminal Region

The gene fragments of nanoKAZ with the deletion of the amino acids atthe amino terminal region were prepared in the following manner.

PCR (cycle conditions: 25 cycles of 1 min/94° C., 1 min/50° C. and 1min/72° C.) was performed with a PCR kit (manufactured by Takara BioInc.) using pcDNA3-GLsp-dnKAZ obtained in EXAMPLE 2 as a temperate withtwo PCR primers.

In preparing, e.g., one amino acid-deleted ΔN2T-nKAZ mutant gene, PCRwas performed using a pcDNA3-GLsp-dnKAZ as a template and the followingtwo primers.

D2-nKAZ-15N/EcoRI  (SEQ ID NO: 23)(5′gccGAATTCAAGC TTGGTACCAC CATGGTCACCCTGGAGG ACTTCGTCGG CGAC 3′:EcoR1 sequence and KpnI sequence underlined)  nanoKAZ-3C/XbaI(SEQ ID NO: 20) (5′gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′)

As a result, the gene (ΔN2T-nKAZ) encoding the amino acid sequence ofnanoKAZ (SEQ ID NO: 2) with the deletion of the phenylalanine residue atthe first amino acid was amplified. The nucleotide sequence of nanoKAZgene is shown by SEQ ID NO: 1. The nucleotide sequence and amino acidsequence of ΔN2T-nKAZ are shown in SEQ ID NO: 3 and SEQ ID NO: 4,respectively.

The nanoKAZ genes deleted at the amino-terminal regions were obtained ina similar manner except for using the templates and primers described inTABLE 2. The amino-terminal sequences of the amino-terminal deletednanoKAZ genes obtained are shown in TABLE 1.

Herein, the nucleotide sequence and amino acid sequence of ΔN3L-nKAZ areshown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The nucleotidesequence and amino acid sequence of ΔN4E-nKAZ are shown in SEQ ID NO: 7and SEQ ID NO: 8, respectively. The nucleotide sequence and amino acidsequence of ΔN5D-nKAZ are shown in SEQ ID NO: 9 and SEQ ID NO: 10,respectively.

TABLE 1 Amino acid sequence of amino-terminal deleted nanoKAZ geneNumber of deleted Deletion amino mutant acids Amino-terminal sequencenanoKAZ 0 FTLEDFVGDWRQTAGYNLDQVLEQG . . . ΔN2T-nKAZ 1 TLEDFVGDWRQTAGYNLDQVLEQG . . . ΔN3L-nKAZ 2  LEDFVGDWRQTAGYNLDQVLEQG . . . ΔN4E-nKAZ 3   EDFVGDWRQTAGYNLDQVLEQG . . . ΔN5D-nKAZ 4    DFVGDWRQTAGYNLDQVLEQG . . . ΔN6F-nKAZ 5     FVGDWRQTAGYNLDQVLEQG . . . ΔN7V-nKAZ 6      VGDWRQTAGYNLDQVLEQG . . . ΔN8G-nKAZ 7       GDWRQTAGYNLDQVLEQG . . . ΔN9D-nKAZ 8        DWRQTAGYNLDQVLEQG . . . ΔN10W-nKAZ 9         WRQTAGYNLDQVLEQG . . . ΔN15G-nKAZ 14              GYNLDQVLEQG . . . ΔN20Q-nKAZ 19                   QVLEQG . . .

TABLE 2 Templates and PCR primers used for deletion of theamino-terminal sequence of nanoKAZ. Deletion mutant Template PrimerSequence ΔN2T-nKAZ pcDNA3-GLsp- a D2-nKAZ-15N/EcoRI 5′ gccGAATCAAGCTTGGTACCAC CATGGTCACCCTGGAGG ACTTCGTCGG dnKAZ CGAC 3′ (SEQ ID NO: 23) bnanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQ IDNO: 20) ΔN3L-nKAZ pcDNA3-GLsp- a D3-nKAZ-16N/ECoRI 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCCTGGAGG dnKAZ ACTTCGTCGG CGACTGG 3′ (SEQ ID NO: 24) bnanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQ IDNO: 20) ΔN4E-nKAZ pcDNA3-GLsp- a D4-nKAZ-17N/ECoRI 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCGAGG dnKAZ ACTTCGTCGG CGACTGGAGA 3′ (SEQ ID NO: 25) bnanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQ IDNO: 20) ΔN5D-nKAZ pcDNA3-GLsp- a D5nanoKAZ-4N/ 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCG ACTTCGTCGG dnKAZ EcoRI CGACTGGAGACAGA 3′ (SEQ ID NO:26) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN6F-nKAZ pcDNA3-GLsp- a D6nanoKAZ-8N/ 5′ gccGAATTCAAGCTTGGTACCAC CATGGTC TTCGTCGG dnKAZ EcoRI CGACTGGAGACAGACC 3′ (SEQ ID NO:27) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN7V-nKAZ pcDNA3-GLsp- a D7nanoKAZ-9N/ 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCGTCGG dnKAZ EcoRI CGACTGGAGACAGACCGCC 3′ (SEQ ID NO:28) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN8G-nKAZ pcDNA3-GLsp- a D8nanoKAZ-10N/ 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCGG dnKAZ EcoRI CGACTGGAGACAGACCGCCG GC 3′ (SEQ ID NO:29) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN9D-nKAZ pcDNA3-GLsp- a D9nanoKAZ-11N/ 5′ gccGAATTCAAGCTTGGTACCAC dnKAZ EcoRI CATGGTCGACTGGAGACAGACCGCCG GCTAC 3′ (SEQ ID NO:30) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN10W-nKAZ pcDNA3-GLsp- a D10nanoKAZ-5N/ 5′ gccGAATTCAAGCTTGGTACCAC dnKAZ EcoRI CATGGTCTGGAGACAGACCGCCG GCTACAACC 3' (SEQ ID NO:31) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′ (SEQID NO: 20) ΔN15G-nKAZ pcDNA3-GLsp- a D15nanoKAZ-6N/ 5′ gccGAATTCAAGCTTGGTACCAC CATGGTCG GCTACAACCT dnKAZ EcoRI GGACCAGGTC CTGG 3′ (SEQ IDNO: 32) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGC ACAGTCT 3′(SEQ ID NO: 20) ΔN20Q-nKAZ pcDNA3-GLsp- a D20nanoKAZ-7N/ 5′gccGAATTCAAGC TTGGTACCAC CATGGTCCAGGTC dnKAZ EcoRI CTGGAGCAGG GCGGCGTCA3′ (SEQ ID NO: 33) b nanoKAZ-3C/XbaI 5′ gccTCTAGA TTAGGCCAGG ATTCTCTCGCACAGTCT 3′ (SEQ ID NO: 20)

Example 4 Construction of E. coli Expression Vectors for Amino-TerminalDeleted nanoKAZ Using a pColdII Vector

The DNA fragment obtained in EXAMPLE 3 was purified with a PCRpurification kit (manufactured by QIAGEN Inc.), digested with therestriction enzymes of EcoRI and XbaI and then ligated to the EcoRI-XbaIsite in the expression vector of pColdII (Takara Bio Inc.) to constructthe expression vectors, pCold-ΔN5D-nKAZ, pCold-ΔN6F-nKAZ,pCold-ΔN7V-nKAZ, pCold-ΔN8G-nKAZ, pCold-ΔN9D-nKAZ and pCold-ΔN10W-nKAZfor the amino-terminal deleted nanoKAZ.

Also, the pCold-ZZ-P-nanoKAZ described in EXAMPLE 1 was digested withthe restriction enzymes of EcoRI and XbaI in a conventional manner andthen ligated to the EcoRI-XbaI site in the expression vector of pColdIIto construct the pCold-nanoKAZ vector. The nucleotide sequence of theinserted DNA was confirmed by sequencing using a DNA Sequencer(manufactured by ABI Inc.). The nucleotide sequence and amino acidsequence of pCold-nanoKAZ are shown in SEQ ID NO: 13 and SEQ ID NO: 14,respectively.

Example 5 Expression of Amino-Terminal Deleted nanoKAZ in E. coli andPreparation of Crude Enzyme Solution

In order to express the amino-terminal deleted nanoKAZ in E. coli, therecombinant plasmid obtained in EXAMPLE 4 was used. The E. coli BL21strain (Novagen, Madison, Wis.) was used as a host cell. The BL21 straincarrying the recombinant plasmid was incubated in 5 mL of Luria-Bertanimedium (hereinafter designated as LB medium) containing ampicillin (50μg/mL) at 37° C. for 18 hours. The seed culture of 0.1 mL was inoculatedto 10 mL of LB medium and incubated for 3 hours, followed by cooling inan ice-water bath for 1 hour. IPTG was added to the culture medium at afinal concentration of 0.1 mM, followed by incubation at 15° C. forfurther 17 hours. After completion of the incubation, 1 mL of theculture medium was collected by centrifugation at 10,000 rpm for 2minutes. The collected E. coli cells were suspended in 0.5 mL of 30 mMTris-HCl (pH 7.6)-10 mM EDTA (manufactured by Wako Pure ChemicalIndustries, Ltd.) (hereinafter designated as TE). The E. coli cells weredisrupted by sonication for 3 seconds using a Branson model 250 sonifire(Danbury, Conn.) to give a crude enzyme solution. Then, 5 μL of thecrude enzyme solution was analyzed by SDS-PAGE to confirm the proteinexpression (FIG. 1).

In FIG. 1, labeled M and 1 to 7 represent as follows: M: molecularweight size markers; 1: nanoKAZ, 2: ΔN5D-nKAZ, 3: ΔN6F-nKAZ, 4:ΔN7V-nKAZ, 5: ΔN8G-nKAZ, 6: ΔN9D-nKAZ and 7: ΔN10W-nKAZ.

Example 6 Construction of E. coli Expression Vectors for ZZ-FusedAmino-Terminal Deleted nanoKAZ

To express the amino-terminal deleted nanoKAZ as a soluble protein, theexpression vector of pCold-ZZ-X (described in Inouye & Sahara, ProteinExpress. Purif. (2009) 66: 52-57) was used. The DNA fragment obtained inEXAMPLE 3 was digested with the restriction enzyme of EcoRI and XbaI andligated to the EcoRI-XbaI site of pCold-ZZ-X to construct the following11 expression vectors for the ZZ-fused amino-terminal deleted nanoKAZ:pCold-ZZ-P-ΔN2T-nKAZ, pCold-ZZ-P-ΔN3L-nKAZ, pCold-ZZ-P-ΔN4E-nKAZ,pCold-ZZ-P-ΔN5D-nKAZ, pCold-ZZ-P-ΔN6F-nKAZ, pCold-ZZ-P-ΔN7V-nKAZ,pCold-ZZ-P-ΔN8G-nKAZ, pCold-ZZ-P-ΔN9D-nKAZ, pCold-ZZ-P-ΔN10W-nKAZ,pCold-ZZ-P-ΔN15G-nKAZ and pCold-ZZ-P-ΔN20Q-nKAZ.

Example 7 Expression of ZZ-Fused Amino-Terminal Deleted nanoKAZ in E.coli and Preparation of Crude Enzyme Solution

To express the ZZ-fused amino-terminal deleted nanoKAZ in E. coli, therecombinant plasmid obtained in EXAMPLE 6 was used. Crude enzymesolutions were prepared using a similar manner to EXAMPLE 5 using the E.coli BL21 strain (Novagen, Madison, Wis.) as a host cell. Then, 5 μL ofthe crude enzyme solution obtained was subjected to SDS-PAGE analysis toconfirm the protein expression (FIG. 2).

In FIG. 2, labeled M and 1 to 12 represent as follows. M: molecularweight size markers; 1: ZZ-P-nanoKAZ, 2: ZZ-P-ΔN2T-nKAZ, 3:ZZ-P-ΔN3L-nKAZ, 4: ZZ-P-ΔN4E-nKAZ, 5: ZZ-P-ΔN5D-nKAZ, 6: ZZ-P-ΔN6F-nKAZ,7: ZZ-P-ΔN7V-nKAZ, 8: ZZ-P-ΔN8G-nKAZ, 9: ZZ-P-ΔN9D-nKAZ, 10:ZZ-P-ΔN10G-nKAZ, 11: ZZ-P-ΔN15G-nKAZ and 12: ZZ-P-ΔN20Q-nKAZ.

Example 8 Assay for Luminescence Activity of Amino-Terminal DeletednanoKAZ and ZZ-Fused Amino-Terminal Deleted nanoKAZ in Crude EnzymeSolution

The crude enzyme solutions obtained in EXAMPLE 5 and EXAMPLE 7 wereallowed to stand in an ice-water for over 1 hour and then diluted to50-fold in TE. By addition of 1 μL each of the crude enzyme solutions to100 μL of TE containing 1 μg of coelenterazine (manufactured by JNCCorp.), a luminescence reaction was started. The luminescence activitywas measured for 60 seconds using a luminometer (manufactured by AttoInc.: AB2200); the maximum intensity of luminescence (I_(max)) is givenas a percentage (%).

The luminescence activities of nanoKAZ, when expressed in E. coli, weremarkedly reduced by deletion of 8 or more amino acid residues at theamino-termius of nanoKAZ. As the deletions up to 5 amino acid resdues atthe amino-terminnus showed 40 to 60% luminescence activity; it was clearthat the amino-terminal region of nanoKAZ had no direct effects on theluminescence activity.

TABLE 3. Luminescence Activity of Amino-Terminal Deleted nanoKAZ inCrude Enzyme Solution

TABLE 3 Relative luminescence activity (%, I_(max)) Deletion mutantpCold- pCold-ZZ-P- nanoKAZ 100 100 ΔN2T-nKAZ — 93.4 ΔN3L-nKAZ — 82.0ΔN4E-nKAZ — 74.0 ΔN5D-nKAZ 87.0 73.6 ΔN6F-nKAZ 40.0 59.8 ΔN7V-nKAZ 1.65.8 ΔN8G-nKAZ 3.9 10 ΔN9D-nKAZ 0.01 0.3 ΔN10W-nKAZ 0.03 1.6 ΔN15G-nKAZ —0 ΔN20Q-nKAZ — 0

Example 9 Secretory Expression Vectors for Amino-Terminal DeletednanoKAZ Mutants Using a Secretory Signal Peptide Sequence of GaussiaLuciferase

From the results of expression of the amino-terminal deleted nanoKAZ inE. coli, detectable luminescence activities were observed in the mutantswith deletions up to 9 amino acid residues from the amino terminus(ΔN10W-nKAZ). In order to confirm that these deletion mutants could besecreted from animal cultured cells, the expression vectors for theamino-terminal deleted nanoKAZ in animal cultured cells wereconstructed.

Specifically, the amino-terminal deleted nanoKAZ gene fragment obtainedin EXAMPLE 3 was digested with the restriction enzymes of EcoRI and XbaIin a conventional manner and then ligated to the EcoRI-XbaI site ofpcDNA3-GLsp obtained in EXAMPLE 2 to construct the expression vectors asfollows; pcDNA3-GLsp-ΔN2T-nKAZ, pcDNA3-GLsp-ΔN3L-nKAZ,pcDNA3-GLsp-ΔN4E-nKAZ, pcDNA3-GLsp-ΔN5D-nKAZ, pcDNA3-GLsp-ΔN6F-nKAZ,pcDNA3-GLsp-ΔN7V-nKAZ, pcDNA3-GLsp-ΔN8G-nKAZ, pcDNA3-GLsp-ΔN9D-nKAZ,pcDNA3-GLsp-ΔN10W-nKAZ and pcDNA3-GLsp-ΔN15G-nKAZ andpcDNA3-GLsp-ΔN20Q-nKAZ.

Also, the pCold-ZZ-P-nanoKAZ vector obtained in EXAMPLE 1 was digestedwith the restriction enzymes of EcoRI and XbaI in a conventional mannerand then ligated to the EcoRI-XbaI site in the expression vector ofpcDNA3-GLsp to construct the pcDNA3-GLsp-nanoKAZ vector.

The gene sequences inserted were confirmed by sequencing using a DNAsequencer (manufactured by ABI Inc.). The nucleotide sequence and aminoacid sequence of pcDNA3-GLsp-nanoKAZ are shown in SEQ ID NO: 15 and SEQID NO: 16, respectively.

Example 10 Transfection of Vectors in Animal Culture Cells andPreparation of Enzyme for Assay

(1) Purification of Expression Plasmid

The following experiment was performed using the recombinant plasmidsobtained in EXAMPLE 9. The recombinant plasmid was purified from E. coliJM83 strain using a plasmid purification kit (manufactured by QIAGEN)and dissolved in sterilized water. The firefly luciferase vector(pGL4.13 [Luc2/sv40]: manufactured by Promega Corp.) was similarlypreapred and used as an internal standard.

(2) Transfection and Preparation of Enzyme for Assay

Chinese hamster ovary cell line CHO-K1 was cultured in Ham's F-12 medium(manufactured by Wako Pure Chemical Industries, Ltd.) supplemented with10% (v/v) fetal bovine serum (manufactured by Biowest Inc.) (hereinaftersometimes referred to as Ham's F-12 medium). The CHO-K1 cells wereseeded in a 6-well plate in 1×10⁵ cells/well/2 mL medium (n=2) andcultured in an incubator at 37° C. in 5% (v/v) CO₂. After 24 hours, thepurified recombinant plasmid was transfected to CHO-K1 cells using aFuGene HD transfection kit (manufactured by Promega Corp.) and the cellswere provided for subsequent experiment. Specifically, 1 μg of therecombinant plasmid, 0.1 μg of the internal standard vector pGL4.13[Luc2/sv40] and 3 μL of FuGene FED were added to 100 μL of the mediumand allowed to stand at room temperature for 15 minutes. Subsequently,100 μL of the DNA-FuGene complex was added to the cells in the 6-wellplate. After incubation for 44 hours, the culture medium was collected.On the other hand, the cells expressed the KAZ mutants were washed 3times with 3 mL of 1×PBS, then suspended in 1 mL of 1×PBS and disruptedby sonication on ice. The resultant cell extracts of nanoKAZ deletionmutants was used as enzyme solutions.

Example 11 Construction of Vectors for the Amino-Terminal DeletednanoKAZ Lacking the Secretory Signal Peptide Sequence in Animal CulturedCells

After digestion of the gene fragment of amino-terminal deleted nanoKAZobtained in EXAMPLE 3 with the restriction enzymes of Asp718 and XbaI,the fragment was inserted into the Asp718-XbaI site of a pcDNA3 vector(manufactured by Invitrogen Inc.) to construct the vectors,pcDNA3-ΔN2T-nKAZ, pcDNA3-ΔN3L-nKAZ, pcDNA3-ΔN4E-nKAZ, pcDNA3-ΔN5D-nKAZ,pcDNA3-ΔN6F-nKAZ, pcDNA3-ΔN7V-nKAZ, pcDNA3-ΔN8G-nKAZ, pcDNA3-ΔN9D-nKAZ,pcDNA3-ΔN10W-nKAZ, pcDNA3-ΔN15G-nKAZ and pcDNA3-ΔN20Q-nKAZ.

Also, the pCold-ZZ-P-nanoKAZ obtained in EXAMPLE 1 was digested with therestriction enzymes of Asp718/XbaI in a conventional manner and thensimilarly ligated to the Asp718-XbaI site of a pcDNA3 vector toconstruct the pcDNA3-nanoKAZ vector. The nucleotide sequence and aminoacid sequence of nanoKAZ in pcDNA3-nanoKAZ are shown in SEQ ID NO: 17and SEQ ID NO: 18, respectively.

Example 12 Transfection of Vectors in Animal Cultured Cells andPreparation of Enzyme for Assay

(1) Purification of Expression Plasmids

The recombinant plasmids obtained in EXAMPLE 11 were purified in asimilar manner to EXAMPLE 10 and dissolved in sterilized water. Thefirefly luciferase vector (pGL4.13 [Luc2/sv40]: manufactured by PromegaCorp.) was similarly prepared and used as an internal standard.

(2) Transfection and Preparation of Enzyme for Assay

Culture media containing the secreted amino-terminal deleted nanoKAZ andcell extracts of amino-terminal deleted nanoKAZ as the enzyme solutionswere prepared in the same manner as in EXAMPLE 10.

Example 13 Assay for Luminescence Activity of Amino-Terminal DeletednanoKAZ Expression in Animal Cultured Cells

After adding of 5 μL each of the culture media and cell extractsobtained in EXAMPLE 10 and EXAMPLE 12, respectively, to 100 μL at of TEcontaining 1 μg of coelenterazine (manufactured by JNC Corp.), aluminescence reaction was started.

The luminescence activity was measured for 60 seconds using aluminometer (manufactured by Atto Inc.: AB2200), and the maximumintensity of luminescence (I_(max)) was given as a percentage (%).

As a result, luminescence activities of the amino-terminal deletednanoKAZ mutants with the secretory signal peptide sequence of Gaussialuciferase were observed in the cytoplasm of ΔN2T-nKAZ to ΔN8G-nKAZ andin the culture media of ΔN2T-nKAZ to ΔN6F-nKAZ.

On the other hand, in the amino-terminal deleted nanoKAZ lacking thesecretory signal peptide sequence, the mutants including those up toΔN5-nKAZ with 4 amino acids deletion from the amino terminus were foundto be secreted into the culture medium, but no secretion from the cellswas observed in the nanoKAZ mutants with the deletion of 5 or more aminoacid residues from the amino terminus. Thus, it became clear that thenanoKAZ mutants with the deletion of at least one to at most 4 aminoacids at the amino terminal region could be secreted extracellularly inthe absence of the secretory signal peptide sequence.

Regarding firefly luciferase used as an internal standard to confirm theefficiency of transfection, 5 μL each of cell extracts obtained inEXAMPLES 10 and 12 were added to 100 μL of a reagent for enzyme assay(manufactured by Promega Corp.) to start a luminescence reaction. Theluminescence activity was measured for 10 seconds using a luminometer(manufactured by Atto Inc.: AB2200). The results reveal that thetransfection efficiencies were almost the same.

TABLE 4 Luminescence activities of amino-terminal deleted nanoKAZ inculture medium and cell extracts Relative luminescence activity (%,I_(max)) pcDNA3-GLsp- pcDNA3- Culture Cell Culture Cell Deletion mutantmedium extracts medium extracts nanoKAZ 100 4.8 100 29.4 ΔN2T-nKAZ 97.03.4 93.4 23.3 ΔN3L-nKAZ 87.7 4.4 50.6 16.2 ΔN4E-nKAZ 60.5 2.9 4.6 9.7ΔN5D-nKAZ 56.2 1.7 0.1 1.5 ΔN6D-nKAZ 0.5 0.3 less than 0.06 0.01ΔN7D-nKAZ 0 0.1 0 less than 0.01 ΔN8D-nKAZ 0 0.2 0 0.01 ΔN9D-nKAZ 0 0 00 ΔN10D-nKAZ 0 0 0 0 ΔN15D-nKAZ 0 0 0 0 ΔN20D-nKAZ 0 0 0 0

From the confirmed results of the activity for the KAZ mutants expressedin E. coli in EXAMPLE 8 and of the secretion from animal cultured cellsin EXAMPLE 13, the nanoKAZ mutants with 1, 2, 3 and 4 amino acidresidues deletion from the amino terminus were secreted into the culturemedium from the animal cultured cells The results reveal that thesequence(s) of at least one to at most 4 amino acid residues from theamino terminus are related to the extracellular secretion of nanoKAZ.

Example 14 Preparation of CHO-K1 Cell Line Stably Expressing nanoKAZHaving the Secretory Signal Peptide Sequence of Gaussia Luciferase

Chinese hamster ovary cell line CHO-K1 was cultured in Ham's F-12medium. The CHO-K1 cells were seeded in a 6 cm petri dish in 2×10⁵cells/well/2 mL medium (n=2) and cultured in an incubator at 37° C. in5% (v/v) CO₂. After 24 hours, the purified pcDNA3-GLsp-nanoKAZ describedin EXAMPLE 10 was transfected to CHO-K1 cells using a FuGene HDtransfection kit (manufactured by Promega Corp.).

The CHO-K1 cells transfected were treated with trypsin and wereresuspended in 5 mL of Ham's F-12 medium. The cell suspension of 0.5×10⁵cells were plated in six petri dishes of 10 cm with 10 mL of Ham's F-12medium. After overnight incubation at 37° C. in a 5% CO₂ incubator, 160μL (a final concentration of 800 μg/mL) of 50 mg/mL G418 sulfate(manufactured by Calbiochem, Inc.) was added thereto, followed byincubation at 37° C. in a 5% CO₂ incubator for 7 days.

Seven days later, 48 colonies formed were picked with a sterilizedtoothpick and suspended in 50 μL of trypsin solution in a 96-well plate.To 50 μL each of the 48 trypsinized-cell suspensions was added 100 μL ofHam's F-12 medium. Each mixture was added to a 24-well plate with 1 mLof Ham's F-12 medium containing G418. The cells were incubated at 37° C.in a 5% CO₂ incubator for about 7 days until the cells grew.

After the cells grew, 5 μL of the culture medium was recovered and addedto 100 μL of TE containing 0.5 μg of coelenterazine, and theluminescence activity was determined. The cell lines showing a highluminescence activity were treated with trypsin and suspended in 1 mL ofHam's F-12 medium containing G418. After 50 μL of the cell suspensionwas added to a 24-well plate with 1 mL of Ham's F-12 medium containingG418, the cells were incubated at 37° C. in a 5% CO₂ incubator for about5 days until the cells grew.

After the cells grew, the luminescence activity was determined in asimilar manner. The cell lines showing a high luminescence activity weretreated with trypsin and suspended in 1 mL of Ham's F-12 mediumcontaining G418. After 200 μL of the cell suspension was added to a6-well plate with 3 mL of Ham's F-12 medium containing G418, the cellswere incubated at 37° C. in a 5% CO₂ incubator for about 5 days untilthe cells grew.

After the cells grew, the luminescence activity was determined in asimilar manner. The cell lines showing a high luminescence activity weretreated with trypsin and suspended in 1 mL of Ham's F-12 mediumcontaining G418 After 500 μL of the cell suspension was added to a 10 cmpetri dish with 10 mL of Ham's F-12 medium containing G418, the cellswere incubated at 37° C. in a 5% CO₂ incubator for approximately 3 to 5days until the cells grew.

After the cells grew, the luminescence activity was determined in asimilar manner to confirm that the cells showed a high luminescenceactivity. The CHO-K1 cell line stably expressing nanoKAZ having asecretory signal peptide sequence of Gaussia luciferase was obtained.

Example 15 Preparation of CHO-K1 Stably Expressing nanoKAZ Lacking theSecretory Signal Peptide Sequence

The CHO-K1 cell line stably expressing nanoKAZ in the absence of thesecretory signal peptide sequence was obtained in a similar manner toEXAMPLE 14, except that pcDNA3-nanoKAZ purified in EXAMPLE 13 as aplasmid DNA for transfection was used.

Example 16 Comparison of Secretory Inhibition by Brefeldin A

Using the stable expressed cell lines established in EXAMPLE 14 andEXAMPLE 15, the effect of brefeldin A that inhibits exocytosis throughthe trans-Golgi network by the vesicle-mediated transport was compared.Specifically, each stable expressed cell lines with 2×10⁵ cells wereseeded to a 6-well plate in 3 mL of Ham's F-12 medium containing 10%FBS, respectively, followed by incubation at 37° C. in a 5% CO₂incubator for 48 hours. After washing twice with 3 mL of Ham's F-12medium containing 10% FBS, brefeldin A (manufactured by Wako PureChemical Industries, Ltd.) was added to the culture medium at a finalconcentration of 5 μg/mL. As a control experiment, cells were culturedin the absence of brefeldin A. The luminescence activity in the culturemedium was determined at each incubation times at 0, 1, 3 and 6 hours,using coelenterazine as a substrate. The results are shown in FIGS. 3and 4.

As shown in FIG. 3, the secretion of nanoKAZ into the culture medium wasmarkedly inhibited by brefeldin A in the pcDNA3-GLsp-nanoKAZ/CHO-K1stable expressed cell line established using the nanoKAZ gene with thesecretory signal peptide sequence, as compared to the cells in theabsence of brefeldin A. This indicates that nanoKAZ having the secretorysignal peptide sequence was secreted normally from a endoplasmicreticulum via the trans-Golgi network.

On the other hand, as shown in FIG. 4, the nanoKAZ was secreted from thepcDNA3-nanoKAZ/CHO-K1 stable expressed cell line established using thenanoKAZ gene without the secretory signal peptide sequence in thepresence or absence of brefeldin A. This indicates that the protein wassecreted through another pathway(s), not through a general trans-Golginetwork from the endoplasmic reticulum.

From the foregoing results and the results of the experiments on theamino-terminal deleted nanoKAZ gene expression described in EXAMPLES 8and 13, it was demonstrated that the amino-terminal region at thepositions of 1 to 4 from the amino terminus of nanoKAZ, has differentsecretion information from a general signal peptide sequence forsecretion.

Example 17 Visualization of nanoKAZ Secretion

Using the stable expressed cell lines established in EXAMPLES 14 and 15,the visualization of nanoKAZ secretion was performed. The 10⁴ cells werecultured in a 35 mm glass bottom plate (manufactured by Iwaki Corp.) in3 mL of MEM-alpha medium (manufactured by Wako Pure Chemical Industries,Ltd.) containing 10% FBS at 37° C. in a 5% CO₂ incubator for 48 hours.After washing 3 times with 3 mL of HBSS (manufactured by Wako PureChemical Industries, Ltd.), h-coelenterazine dissolved in HBSS at afinal concentration of 3 μg/mL was added to cells. The visualized imageswere captured using an IX81-ZDC microscope (manufactured by OlympusOptical Co.) equipped with an EM-CCD camera (ImagEM 1K: manufactured byHamamatsu Photonics K.K.) under image acquisition conditions (1×1binning, fast scanning, EM-gain level 255, photon-counting level=1,image acquisition time, 0.5 sec.). After the addition ofh-coelenterazine in 15 seconds, the captured images are shown in FIG. 5.Labeled a and b designate the bright-field image and the luminescenceimage of nanoKAZ from pcDNA3-GLsp-nanoKAZ/CHO-K1 stable expressed cells,respectively. Labeled c and d designate the bright-field image and theluminescence image of nanoKAZ from pcDNA3-nanoKAZ/CHO-K1 stableexpression cells, respectively. Comparison between the luminescenceimages b and d obtained by the luminescence imaging indicate that,unlike nanoKAZ from the pcDNA3-GLsp-nanoKAZ/CHO-K1 cells secreted fromthe endoplasmic reticulum through the trans-Golgi network, nanaoKAZ fromthe pcDNA3-nanoKAZ/CHO-K1 cells were uniformly distributed on the cellsurface and were secreted outside of cells, showing a different mode ofsecretion. More specifically, based on the results of experiments ofamino-terminal deleted nanoKAZ gene expression in EXAMPLES 8 and 13, itwas revealed that different secretion information from that of general.secretory signal peptide sequences was found in the amino acid region atthe positions of 1 to 4 from the amino terminus of nanoKAZ and,therefore, the visualized images of secretion were different as well.

Example 18 Substrate Specificities for Amino-Terminal Deleted nanoKAZ

The enzyme solutions of amino-terminal deleted nanoKAZ used forsubstrate specificity studies were prepared by the method described inEXAMPLE 12, using pcDNA3 vectors (pcDNA3-ΔN2T-nKAZ, pcDNA3-ΔN3L-nKAZ,pcDNA3-ΔN4E-nKAZ, pcDNA3-ΔN5D-nKAZ and pcDNA3-ΔN6F-nKAZ) for expressionof the amino-terminal deletions, which were obtained by the methoddescribed in EXAMPLE 11. After transfection to CHO-K1 cells and thenincubation for 48 hours, the culture medium were collected and stored ina frozen state. The coelenterazine analogues used for substratespecificity studies were synthesized by the methods described inpublications, respectively. Specifically, coelenterazine (CTZ),h-coelenterazine (h-CTZ) and f-coelenterazine (f-CTZ),6h-f-coelenterazine (6h-f-CTZ) were synthesized by the method describedin Inouye et al (2013) Biochem. Biophys. Res. Commun. 437: 23-28, andbis-coelenterazine (bis-CTZ) was synthesized by the method described inNakamura et al. (1997) Tetrahedron Lett. 38: 6405-6406.

A luminescence reaction was started by the addition of 2 μL of thefreeze-thawed culture medium on ice to 100 μL of TE containing 1 μg ofcoelenterazine or its analogue. The luminescence activities weremeasured for 10 seconds using a luminometer (manufactured by Atto Inc.:AB2200), and the maximum intensity of luminescence (I_(max)) of nanoKAZwas shown as a relative luminescence activity using coelenterazine as asubstrate. The results are shown in TABLE 5.

TABLE 5 Relative luminescence activity (I_(max)) Deletion mutant CTZbis-CTZ 6h-f-CTZ h-CTZ f-CTZ nanoKAZ 1.00 6.93 7.00 11.76 12.65ΔN2T-nKAZ 1.03 8.99 8.02 13.38 15.04 ΔN3L-nKAZ 0.50 5.39 5.25 9.39 9.88ΔN4E-nKAZ 0.05 0.35 0.36 0.58 0.70 ΔN5D-nKAZ 0.01 0.01 0.01 0.01 0.02ΔN6F-nKAZ less than less than less than less than less than 0.01 0.010.01 0.01 0.01

As shown in TABLE 5, when bis-CTZ, 6h-f-CTZ, h-CTZ and f-CTZ were usedas substrates, the nanoKAZ deletion mutants of ΔN2T-nKAZ, ΔN3L-nKAZ andΔN4E-nKAZ showed approximately 8 to 15-fold higher activity thancoelenterazine (CTZ) used as the substrate. Particularly, h-CTZ andf-CTZ.showed over 10-fold higher activity than coeleneterazine. Inaddition, the nanoKAZ deletion mutant of ΔN2T-nKAZ was found to show ahigher activity than nanoKAZ when bis-CTZ, 6h-f-CTZ, h-CTZ and f-CTZwere used.

Sequence Listing Free Text

-   [SEQ ID NO: 1] Nucleotide sequence of nanoKAZ-   [SEQ ID NO: 2] Amino acid sequence of nanoKAZ-   [SEQ ID NO: 3] Nucleotide sequence of ΔN2T-nKAZ-   [SEQ ID NO: 4] Amino acid sequence of ΔN2T-nKAZ-   [SEQ ID NO: 5] Nucleotide sequence of ΔN3L-nKAZ-   [SEQ ID NO: 6] Amino acid sequence of ΔN3L-nKAZ-   [SEQ ID NO: 7] Nucleotide sequence of ΔN4E-nKAZ-   [SEQ ID NO: 8] Amino acid sequence of ΔN4E-nKAZ-   [SEQ ID NO: 9] Nucleotide sequence of ΔN5D-nKAZ-   [SEQ ID NO: 10] Amino acid sequence of ΔN5D-nK-   [SEQ ID NO: 11] Nucleotide sequence of pCold-ZZ-P-nanoKAZ-   [SEQ ID NO: 12] Amino acid sequence of pCold-ZZ-P-nanoKAZ-   [SEQ ID NO: 13] Nucleotide sequence of pCold-nanoKAZ-   [SEQ ID NO: 14] Amino acid sequence of pCold-nanoKAZ-   [SEQ ID NO: 15] Nucleotide sequence of pcDNA3-GLsp-nanoKAZ-   [SEQ ID NO: 16] Amino acid sequence of pcDNA3-GLsp-nanoKAZ-   [SEQ ID NO: 17] Nucleotide sequence of pcDNA3-nanoKAZ-   [SEQ ID NO: 18] Amino acid sequence of pcDNA3-nanoKAZ-   [SEQ ID NO: 19] Nucleotide sequence of the primer used in EXAMPLES    (nanoKAZ-1N/EcoRI)-   [SEQ ID NO: 20] Nucleotide sequence of the primer used in EXAMPLES    (nanoKAZ-3C/XbaI)-   [SEQ ID NO: 21] Nucleotide sequence of the primer used in EXAMPLES    (GLsp-1R/EcoRI)-   [SEQ ID NO: 22] Nucleotide sequence of the primer used in EXAMPLES    (T7 primer)-   [SEQ ID NO: 23] Nucleotide sequence of the primer used in EXAMPLES    (D2-nKAZ-15N/EcoRI)-   [SEQ ID NO: 24] Nucleotide sequence of the primer used in EXAMPLES    (D3-nKAZ-16N/ECoRI)-   [SEQ ID NO: 25] Nucleotide sequence of the primer used in EXAMPLES    (D4-nKAZ-17N/ECoRI)-   [SEQ ID NO: 26] Nucleotide sequence of the primer used in EXAMPLES    (D5nanoKAZ-4N/EcoRI)-   [SEQ ID NO: 27] Nucleotide sequence of the primer used in EXAMPLES    (D6nanoKAZ-8N/EcoRI)-   [SEQ ID NO: 28] Nucleotide sequence of the primer used in EXAMPLES    (D7nanoKAZ-9N/EcoRI)-   [SEQ ID NO: 29] Nucleotide sequence of the primer used in EXAMPLES    (D8nanoKAZ-10N/EcoRI)-   [SEQ ID NO: 30] Nucleotide sequence (D9nanoKAZ-11N/EcoRI) of the    primer used in EXAMPLES-   [SEQ ID NO: 31] Nucleotide sequence of the primer used in EXAMPLES    (D10nanoKAZ-5N/EcoRI)-   [SEQ ID NO: 32] Nucleotide sequence of the primer used in EXAMPLES    (D15nanoKAZ-6N/EcoRI)-   [SEQ ID NO: 33] Nucleotide sequence of the primer used in EXAMPLES    (D20nanoKAZ-7N/EcoRI)

The invention claimed is:
 1. A method for performing a luminescencereaction, which comprises contacting a luciferase mutant with aluciferin, wherein the luciferase mutant is selected from (a) to (e)below: (a) a luciferase mutant consisting of an amino acid sequence inwhich at least one amino acid selected from amino acids at the positionsof 1 to 4 is deleted in the amino acid sequence of SEQ ID NO: 2; (b) aluciferase mutant consisting of an amino acid sequence in which at leastone amino acid selected from amino acids at the positions of 1 to 4 isdeleted in the amino acid sequence of SEQ ID NO: 2 and an amino acidsequence excluding the amino acids at the positions of 1 to 4 is anamino acid sequence in which 1 to 17 amino acids are deleted,substituted, inserted and/or added in the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;(c) a luciferase mutant consisting of an amino acid sequence in which atleast one amino acid selected from amino acids at the positions of 1 to4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 hasat least 90% identity to the amino acid sequence at the positions of 5to 169 of SEQ ID NO: 2, and having a luciferase activity; (d) aluciferase mutant consisting of an amino acid sequence in which at leastone amino acid selected from amino acids at the positions of 1 to 4 isdeleted in the amino acid sequence of SEQ ID NO: 2 and an amino acidsequence excluding the amino acids at the positions of 1 to 4 is encodedby a polynucleotide which hybridizes under high stringent conditions toa polynucleotide consisting of a nucleotide sequence complementary to anucleotide sequence encoding the amino acid sequence at the positions of5 to 169 of SEQ ID NO: 2, wherein the high stringent conditions are5×SSC, 5×Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50°C., and having a luciferase activity; and, (e) a luciferase mutantconsisting of an amino acid sequence selected from SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8 and SEQ ID NO:
 10. 2. The method according to claim1, wherein the luciferin is coelenterazines.
 3. The method according toclaim 2, wherein the coelenterazines is bis-coelenterazine or6h-f-coelenterazine.
 4. A method for visualizing a luminescencereaction, which comprises contacting a luciferase mutant with aluciferin, and capturing a visualized image wherein the luciferasemutant is selected from (a) to (e) below: (a) a luciferase mutantconsisting of an amino acid sequence in which at least one amino acidselected from amino acids at the positions of 1 to 4 is deleted in theamino acid sequence of SEQ ID NO: 2; (b) a luciferase mutant consistingof an amino acid sequence in which at least one amino acid selected fromamino acids at the positions of 1 to 4 is deleted in the amino acidsequence of SEQ ID NO: 2 and an amino acid sequence excluding the aminoacids at the positions of 1 to 4 is an amino acid sequence in which 1 to17 amino acids are deleted, substituted, inserted and/or added in theamino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2, andhaving a luciferase activity; (c) a luciferase mutant consisting of anamino acid sequence in which at least one amino acid selected from aminoacids at the positions of 1 to 4 is deleted in the amino acid sequenceof SEQ ID NO: 2 and an amino acid sequence excluding the amino acids atthe positions of 1 to 4 has at least 90% identity to the amino acidsequence at the positions of 5 to 169 of SEQ ID NO: 2, and having aluciferase activity; (d) a luciferase mutant consisting of an amino acidsequence in which at least one amino acid selected from amino acids atthe positions of 1 to 4 is deleted in the amino acid sequence of SEQ IDNO: 2 and an amino acid sequence excluding the amino acids at thepositions of 1 to 4 is encoded by a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to a nucleotide sequence encoding theamino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2,wherein the high stringent conditions are 5×SSC, 5×Denhart's solution,0.5% (w/v) SDS, 50% (v/v) formamide and 50° C., and having a luciferaseactivity; and, (e) a luciferase mutant consisting of an amino acidsequence selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQID NO:
 10. 5. The method according to claim 4, wherein the luciferin iscoelenterazines.
 6. The method according to claim 5, wherein thecoelenterazines is h-coelenterazine or f-coelenterazine.
 7. The methodaccording to claim 1, wherein the luciferase mutants defined in (b) to(d) above are mutants defined in (b-1) to (d-1) below: (b-1) aluciferase mutant consisting of an amino acid sequence in which at leastone amino acid selected from amino acids at the positions of 1 to 4 isdeleted in the amino acid sequence of SEQ ID NO: 2 and an amino acidsequence excluding the amino acids at the positions of 1 to 4 is anamino acid sequence in which 1 to 9 amino acids are deleted,substituted, inserted and/or added in the amino acid sequence at thepositions of 5 to 169 of SEQ ID NO: 2, and having a luciferase activity;(c-1) a luciferase mutant consisting of an amino acid sequence in whichat least one amino acid selected from amino acids at the positions of 1to 4 is deleted in the amino acid sequence of SEQ ID NO: 2 and an aminoacid sequence excluding the amino acids at the positions of 1 to 4 hasat least 95% identity to the amino acid sequence at the positions of 5to 169 of SEQ ID NO: 2, and having a luciferase activity; and, (d-1) aluciferase mutant consisting of an amino acid sequence in which at leastone amino acid selected from amino acids at the positions of 1 to 4 isdeleted in the amino acid sequence of SEQ ID NO: 2 and an amino acidsequence excluding the amino acids at the positions of 1 to 4 is encodedby a polynucleotide which hybridizes under high stringent conditions toa polynucleotide consisting of a nucleotide sequence complementary to anucleotide sequence encoding the amino acid sequence at the positions of5 to 169 of SEQ ID NO: 2, wherein the high stringent conditions are5×SSC, 5×Denhart's solution, 0.5% (w/v) SDS, 50% (v/v) formamide and 50°C., and having a luciferase activity.
 8. The method according to claim4, wherein the luciferase mutants defined in (b) to (d) above aremutants defined in (b-1) to (d-1) below: (b-1) a luciferase mutantconsisting of an amino acid sequence in which at least one amino acidselected from amino acids at the positions of 1 to 4 is deleted in theamino acid sequence of SEQ ID NO: 2 and an amino acid sequence excludingthe amino acids at the positions of 1 to 4 is an amino acid sequence inwhich 1 to 9 amino acids are deleted, substituted, inserted and/or addedin the amino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2,and having a luciferase activity; (c-1) a luciferase mutant consistingof an amino acid sequence in which at least one amino acid selected fromamino acids at the positions of 1 to 4 is deleted in the amino acidsequence of SEQ ID NO: 2 and an amino acid sequence excluding the aminoacids at the positions of 1 to 4 has at least 95% identity to the aminoacid sequence at the positions of 5 to 169 of SEQ ID NO: 2, and having aluciferase activity; and, (d-1) a luciferase mutant consisting of anamino acid sequence in which at least one amino acid selected from aminoacids at the positions of 1 to 4 is deleted in the amino acid sequenceof SEQ ID NO: 2 and an amino acid sequence excluding the amino acids atthe positions of 1 to 4 is encoded by a polynucleotide which hybridizesunder high stringent conditions to a polynucleotide consisting of anucleotide sequence complementary to a nucleotide sequence encoding theamino acid sequence at the positions of 5 to 169 of SEQ ID NO: 2,wherein the high stringent conditions are 5×SSC, 5×Denhart's solution,0.5% (w/v) SDS, 50% (v/v) formamide and 50° C., and having a luciferaseactivity.